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Historically treated as monomeric polypeptides, G protein-coupled receptors (GPCRs) have been shown to exist and function as constitutively formed dimers or oligomers. The quaternary structure of GPCRs may modulate ligand binding properties through allosteric mechanisms offering new opportunities for drug design by exploiting multivalency. In this context, multivalent ligands versus bivalent-ligands, possessing two binding motifs connected by a linker, have been investigated and have revealed striking differences in their functional properties compared to their monovalent counterparts. These bi-functional drugs, which are able to activate the two protomers in a dimer simultaneously, emerge as novel and promising drugs for a variety of multi-factorial diseases. In this review, key requirements for the successful design and synthesis of GPCR multivalent ligands composed of pharmacophores and a linker will be discussed. We will then focus on the 5-HT(4) receptor (5-HT(4)R), whose ligands emerged as promising drugs for a variety of central nervous disorders. Upon description of biochemical and biophysical evidences of 5-HT(4)R dimerization, we will present the multivalent ligand approach, which was assisted by molecular docking experiments on the 5-HT(4)R dimer model.

Background: The serotonin 5-HT(4) receptor is of potential interest for the treatment of Alzheimer's disease because it increases memory and learning and influences amyloid precursor protein (APP) processing. Several 5-HT(4) receptor isoforms have been cloned in humans. They all belong to the G-protein-coupled receptor superfamily and differ in composition and length of their amino acid intracellular C-terminal sequence. At present, it is still unknown whether 5-HT(4) receptor isoforms may have distinct effects on APP processing., Objective: In this study, we investigated the effect of an ultrashort isoform of the human 5-HT(4) receptor (2 amino acids after the splicing site), the 5-HT(4(d)) receptor isoform (h5-HT(4(d))), on APP processing in Chinese hamster ovary cells (CHO cells)., Methods: Non-amyloidogenic soluble sAPPalpha was determined by immunoblot and beta-amyloid peptides (Abeta(1-40) and Abeta(1-42)) were assayed by ELISA in CHO cells stably expressing h5-HT(4(d)) and transiently transfected with human APP(695)., Results: We show here that activation of h5-HT(4(d)) strongly stimulates the release of sAPPalpha and concomitantly decreases extracellular Abeta peptides. These data contrast with our previous findings showing that the h5-HT(4(e/g)) receptor isoform, which has 20 amino acids after the splicing site, did not influence Abeta secretion., Conclusion: We conclude that C-terminal tails of 5-HT(4) receptor isoforms may influence their functional effect on Abeta secretion and that the pathways regulating either beta- or gamma-secretase may be differentially controlled by 5-HT(4) receptor isoforms., (2008 S. Karger AG, Basel)

G-protein-coupled receptor dimerization directs the design of new drugs that specifically bind to receptor dimers. Here, we generated a targeted series of homobivalent ligands for serotonin 5-HT(4) receptor (5-HT(4)R) dimers composed of two 5-HT(4)R-specific ML10302 units linked by a spacer. The design of spacers was assisted by molecular modeling using our previously described 5-HT(4)R dimer model. Their syntheses were based on Sonogashira-Linstrumelle coupling methods. All compounds retained high-affinity binding to 5-HT(4)R but lost the agonistic character of the monomeric ML10302 compound. Direct evidence for the functional interaction of both pharmacophores of bivalent ligands with the 5-HT(4)R was obtained using a bioluminescence resonance energy transfer (BRET) based assay that monitors conformational changes within 5-HT(4) dimers. Whereas the monovalent ML10302 was inactive in this assay, several bivalent derivatives dose-dependently increased the BRET signal, indicating that both pharmacophores functionally interact with the 5-HT(4) dimer. These bivalent ligands may serve as a new basis for the synthesis of potential drugs for 5-HT(4)-associated disorders.

Recent evidence suggests that 5-hydroxytryptamine (5-HT)(4) receptor activity enhances cognition and provides neuroprotection. Here we report the effects of VRX-03011, a novel partial 5-HT(4) agonist, that is both potent (K(i) approximately 30 nM) and highly selective (K(i) > 5 microM for all other 5-HT receptors tested). In separate experiments, rats received VRX-03011 (0.1-10 mg/kg i.p.) 30 min prior to spontaneous alternation testing in a no-delay or a 30-s delay condition. VRX-03011 (1, 5 and 10 mg/kg, but not 0.1 mg/kg) significantly enhanced delayed spontaneous alternation performance while none of the doses enhanced performance in the no-delay test. VRX-03011 (1 and 5 mg/kg) concomitantly enhanced hippocampal acetylcholine output and delayed spontaneous alternation scores compared to that of vehicle controls, but had no effect on hippocampal acetylcholine release under a resting condition. Moreover, suboptimal doses of VRX-03011 and the acetylcholinesterase inhibitor galanthamine combined to enhance memory. VRX-03011 also regulated amyloid precursor protein (APP) metabolism by inducing a concentration-dependent increase in the non-amyloidogenic soluble form of APP (sAPPalpha) with an EC(50) approximately 1--10 nM. VRX-03011 had no effect on contractile properties in guinea pig ileum or colon preparations with an EC(50) > 10 microM and did not alter rat intestinal transit at doses up to 10 mg/kg. These findings suggest that VRX-03011 may represent a novel treatment for Alzheimer's disease that reduces cognitive impairments and provides neuroprotection without gastrointestinal side effects.

Serotonin 5-HT(4) receptors are present in human atrial myocytes and have been proposed to contribute to the generation of atrial fibrillation (AF). Here, we quantified 5-HT(4) receptors as well as other key genes involved in cardiac rhythm and contraction in right atrial appendages of patients with chronic AF (CAF) and acute AF (AAF). Right atrial appendages were obtained from eleven patients in sinus rhythm (SR), five with AAF and six with CAF (>12 months). TaqMan real time quantitative RT-PCR was performed on total RNA. Results were normalised to the average of three housekeeping genes, cyclophilin, GADPH and RL-19. The rank order of expression of h5-HT(4) receptors variants was (b)>(a)>(g)>(c) in the group of patients in SR. In AAF, we found a strong decrease in h5-HT(4(b)), h5-HT(4(c),) and h5-HT(4(g)) transcripts. In CAF patients, the mRNA expression level of the h5-HT(4(b)) isoform significantly increased two fold versus SR. A similar increase was reported for beta(1)-adrenergic receptor, connexin 43 and the L-type Ca(2+) channel CaCNA1C subunit. Interestingly, CAF was associated with a strong increase in the expression of Na(+)/Ca(2+) exchanger and the voltage-dependent Na(+) channel SCN5A subunit. Our results indicate that h5-HT(4(b)) is the dominant cardiac isoform of human 5-HT(4) receptors and its expression is increased in CAF. These data support the involvement of 5-HT(4) receptors in atrial arrhythmia.

The 5-HT(4) receptor (5-HT(4)R) belongs to the G-protein-coupled receptor (GPCR) family and is of considerable interest for the development of new drugs to treat gastrointestinal diseases and memory disorders. The 5-HT(4)R exists as a constitutive dimer but its molecular determinants are still unknown. Using co-immunoprecipitation and Bioluminescence Resonance Energy Transfer (BRET) techniques, we show here that 5-HT(4)R homodimerization but not 5-HT(4)R-beta(2) adrenergic receptor (beta(2)AR) heterodimerization is largely decreased under reducing conditions suggesting the participation of disulfide bonds in 5-HT(4)R dimerization. Molecular modeling and protein docking experiments identified four cysteine (Cys) residues potentially involved in the dimer interface through intramolecular or intermolecular disulfide bonds. We show that disulfide bridges between Cys112 and Cys145 located within TM3 and TM4, respectively, are of critical importance for 5-HT(4)R dimer formation. Our data suggest that two disulfide bridges between two transmembrane Cys residues are involved in the dimerization interface of a GPCR.

Background: The presence of functional 5-HT4 receptors in human and its involvement in neonatal lupus erythematosus (NLE) have prompted us to study the receptor expression and role during embryogenesis. Earlier we managed to demonstrate that female BALB/c mice immunized against the second extracellular loop (SEL) of the 5-HT4 receptor gave birth to pups with heart block. To explain this phenomenon we investigated the expression of 5-HT4 receptors during mouse embryogenesis. At the same time we looked whether the consequence of 5-HT4 receptor immunomodulation observed earlier is in relation to receptor expression. We studied the expression of 5-HT4 receptor at the mRNA level and its two isoforms 5-HT4(a) and 5-HT4(d) at the protein level in embryos from BALB/c mice, at 8th, 12th, 18th gestation days (GD) and 1 day post natal (DPN). Simultaneously the receptor activity was inhibited by rising antibodies, in female mice against SEL of the receptor. The mice were mated and embryos were collected at 8th, 12th, 18th GD and 1 DPN., Results: 5-HT4 receptor mRNA increased in brain from 12th GD to 1 DPN. Its expression gradually decreased in heart and disappeared at birth. This was consistent with expression of the receptor isoforms 5-HT4(a) and (d). Abnormalities like decreased number of embryos, growth delay, spina bifida and sinus arrhythmia from 12th GD were documented in pups of mice showing anti-5-HT4 receptor antibodies., Conclusion: serotoninergic 5-HT4 receptor plays an important role in mouse foetal development. In BALB/c mice there is a direct relation between the expression of receptor and the deleterious effect of maternal anti-5-HT4 receptor autoantibodies in early embryogenesis.

Recently, human 5-HT4 receptors have been demonstrated to form constitutive dimers in living cells. To evaluate the role of dimerization on the 5-HT4 receptor function, we investigated the conception and the synthesis of bivalent molecules able to influence the dimerization process. Their conception is based on a model of the 5-HT4 receptor dimer derived from protein/protein docking experiments. These bivalent ligands are constituted by two ML10302 units, a specific 5-HT4 ligand, linked through a spacer of different sizes and natures. These synthesized bivalent ligands were evaluated in binding assays and cyclic AMP production on the 5-HT4(e/g) receptor isoform stably transfected in C6 glial cells. Our data showed that bivalent ligands conserved a similar affinity compared to the basal ML10302 unit. Nevertheless, according to the nature and the size of the spacer, the pharmacological profile of ML10302 is more or less conserved. In view of the interest of bivalent ligands for investigating the GPCR dimerization process, these 5-HT4 specific bivalent ligands constitute valuable pharmacological tools for the study of 5-HT4 receptor dimerization.

Splenocytes from a BALB/c mouse immunised with a synthetic peptide corresponding to the second extracellular loop of the 5-HT4 receptor were fused with SP2/O myeloma cells to produce a monoclonal antibody. The monoclonal antibody was of the IgG2b isotype. The antibody recognised the human 5-HT4(g) (h5-HT4(g)) receptor by immunoblots and by immunofluorescence on chinese hamster ovary (CHO) cells expressing this 5-HT4 receptor isoform. Epitope mapping of the antibody suggested the recognition of a conformational epitope, encompassing the N- and C-terminal fragments of the second extracellular loop. Kinetic experiments using surface plasmon resonance showed that the antibody had a picomolar affinity for its cognate peptide. Inhibition experiments using the same methodology confirmed the specificity of the interaction. The antibody at a concentration of 500 pM competitively inhibited inverse agonist GR113808 binding and showed an inverse agonist effect on the basal activity of CHO cells expressing the 5-HT4(g) receptor. The antibody decreased the effect of 5-HT at 500 and 50 pM concentrations but it increased 5-HT-induced cAMP levels at 5 pM. The dual effect of the monoclonal antibody could be ascribed to mono- or bivalent recognition of the receptor. The antibody described here is the first example of a high-affinity modulator of the 5-HT4 receptor.

Serotonin 5-HT4 receptors are present in human atrial myocytes and have been proposed to contribute to the generation of atrial fibrillation. However, 5-HT4 receptors have so far been only found in human and pig atria and are absent from the heart of small laboratory animals, such as rat, guinea pig, rabbit and frog, which limits the experimental settings for studying their functional properties. In this study, we developed an adenovirus expression system to examine the properties of two human 5-HT4 receptor splice variants, h5-HT4(b) and h5-HT4(d), expressed in adult cardiomyocytes devoid of native 5-HT4 receptors. When expressed in the HL-1 murine cell line of atrial origin, both receptors caused specific binding of the 5-HT4 selective antagonist GR113808 and activated adenylyl cyclase in the presence of serotonin (5-HT, 1 microM). When expressed in freshly isolated adult rat ventricular cardiomyocytes, a stimulation of the L-type Ca2+ current (ICa,L) by 5-HT (100 nM) was revealed. Both effects were blocked by GR113808. In HL-1 cells, the h5-HT4(d) receptor was found to be more efficiently coupled to adenylyl cyclase than the h5-HT4(b). Pertussis toxin treatment (250 ng/ml for 5 h) potentiated the stimulatory effect of 5-HT on ICa,L in rat myocytes expressing the h5-HT4(b) but not the h5-HT4(d) receptor, indicating a likely coupling of the (b) isoform to both Gs and Gi/o proteins. Adenoviral expression of h5-HT4 receptor isoforms in adult cardiac myocytes provides a valuable means for the exploration of the receptor signaling cascades in normal and pathological situations.

The serotonin 5-HT4 receptor (where 5-HT stands for 5-hydroxy-tryptamine) is a member of the seven transmembrane-spanning G-protein-coupled family of receptors and mediates many cellular functions both in the central nervous system and at the periphery. In the present study, we isolated and characterized the 5'-flanking region of the h5-HT4 (human 5-HT4) receptor. We demonstrate the existence of a novel exon that corresponds to the 5'-untranslated region of the h5-HT4 receptor gene. RNase protection analysis and reverse transcriptase-PCR experiments performed on human atrial RNA demonstrated that the major transcription start site of the h5-HT4 receptor gene is located at -3185 bp relative to the first ATG codon. In addition, a 1.2 kb promoter fragment which drives the transcription of the 5-HT4 receptor was characterized. The promoter region lacks TATA and CAAT canonical motifs in the appropriate location, but contains putative binding sites for several transcription factors. Transient transfection assays revealed that the (-3299/-3050) gene fragment possesses the ability to promote the expression of the luciferase reporter gene in human cell lines. In contrast, the promoter was silent in monkey COS-7 cells, indicating the requirement of specific factors to initiate transcription in human cells. In addition to the promoter element, enhancer activity was found in a region (-220/-61) located in the long 5'-untranslated region. Mutational analysis, gel shift and transfection assays identified an Nkx2.5 (NK2-transcription-factor-related 5)-like binding site as a regulatory sequence of this enhancer. Our results suggest a complex regulation of the h5-HT4 receptor gene expression involving distinct promoters and non-coding exons.

The serotonin 5-hydroxytryptamine (5-HT4) receptor is of potential interest for the treatment of Alzheimer's disease because it increases memory and learning. In this study, we investigated the effect of zinc metalloprotease inhibitors on the amyloid precursor protein (APP) processing induced by the serotonin 5-HT4 receptor in vitro. We show that secretion of the non-amyloidogenic form of APP, sAPPalpha induced by the 5-HT4(e) receptor isoform was not due to a general boost of the constitutive secretory pathway but rather to its specific effect on alpha-secretase activity. Although the h5-HT4(e) receptor increased IP3 production, inhibition of PKC did not modify its effect on sAPPalpha secretion. In addition, we found that alpha secretase activity is regulated by the cAMP-regulated guanine nucleotide exchange factor, Epac and the small GTPase Rac.

A body of evidences suggests that a hydrophobic pocket of the human 5-HT(4) receptor contributes to the high affinity of some bulky 5-HT(4) ligands. A thorough study of this pocket was performed using mutagenesis and molecular modeling. Ligand binding or competition studies with selected bulky ligands (RS39604, RS100235, [(3)H]GR113808 and ML11411) and small ligands (5-HT and ML10375) were carried out on wild-type and mutant receptors (W7.40A/F, Y7.43F, R3.28L) transiently transfected in COS-7 cells. The functional activity of the mutated receptors was evaluated by measuring the ability of 5-HT to stimulate adenylyl cyclase. For W7.40F mutation, no changes in the affinity of studied ligands and in the functional activity of the mutant receptor were observed, in contrary to W7.40A mutation, which abolished both binding of ligands and 5-HT-induced cAMP production. Mutation R3.28L revealed a totally silent receptor with a basal level of cAMP production similar to the mock control despite its ability to product cAMP in the presence of 5-HT. Moreover, a one order loss of affinity of RS39604 and a 45-fold increase of ML11411 affinity were observed. Mutation Y7.43F modified the affinity of GR113808, which displays a 13-fold lower affinity for the mutant than for the wild-type receptor. In conclusion, in the hydrophobic pocket, two polar amino acids are able to interact through hydrogen bonds with bulky ligands depending on their chemical properties. Moreover, these experimental data may validate the proposed new three-dimensional model of the human 5-HT(4) receptor.

The serotonin 5-HT4 receptor mediates many physiological effects in the central nervous system. The recent molecular identification of 5-HT4 receptors and the development of selective 5-HT4 receptor ligands have led to many important new insights into the signalling pathways and the physiological roles of these G protein-coupled-receptors in neurones. With respect to neurodegenerative disorders, it is suggested that 5-HT4 agonists may represent a new avenue for the treatment of Alzheimer's disease. This mini-review will focus on recent in vitro and in vivo pharmacological and biochemical studies showing the involvement of 5-HT4 receptors in cognitive processes and the amyloid precursor protein processing. The potential use of 5-HT4 agonists for the treatment of AD will be discussed.