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A Single Mutation in the 5-HT4 Receptor (5-HT4-R D100(3.32)A) Generates a Gs-coupled Receptor Activated Exclusively by Synthetic Ligands (RASSL)

2003; Elsevier BV; Volume: 278; Issue: 2 Linguagem: Inglês

10.1074/jbc.c200588200

1083-351X

Sylvie Claeysen, Lara Joubert, Michèle Sebben, Joël Bockaert, Aline Dumuis,

Chemical Synthesis and Analysis

To better understand G-protein-coupled receptor (GPCRs) signaling, cellular and animal physiology, as well as gene therapy, a new tool has recently been proposed. It consists of GPCR mutants that are insensitive to endogenous ligands but sensitive to synthetic ligands. These GPCRs are called receptor activated solely by synthetic ligands (RASSL). Only two examples of such engineered receptors have been described so far: one Gi-coupled (opioid receptors) and one Gs-coupled (β2-adrenergic receptors). Here, we describe the first RASSL related to serotonin receptors (D100(3.32)A Gs-coupled 5-HT4 receptor or 5-HT4-RASSL). 5-HT4-RASSL is generated by a single mutation, is totally insensitive to serotonin (5-HT), and still responds to synthetic ligands. These ligands have affinities in the range of nanomolar concentrations for the mutant receptor and exhibit full efficacy. More interestingly, two synthetic ligands behave as antagonists on the wild type but as agonists on the 5-HT4-RASSL. To better understand G-protein-coupled receptor (GPCRs) signaling, cellular and animal physiology, as well as gene therapy, a new tool has recently been proposed. It consists of GPCR mutants that are insensitive to endogenous ligands but sensitive to synthetic ligands. These GPCRs are called receptor activated solely by synthetic ligands (RASSL). Only two examples of such engineered receptors have been described so far: one Gi-coupled (opioid receptors) and one Gs-coupled (β2-adrenergic receptors). Here, we describe the first RASSL related to serotonin receptors (D100(3.32)A Gs-coupled 5-HT4 receptor or 5-HT4-RASSL). 5-HT4-RASSL is generated by a single mutation, is totally insensitive to serotonin (5-HT), and still responds to synthetic ligands. These ligands have affinities in the range of nanomolar concentrations for the mutant receptor and exhibit full efficacy. More interestingly, two synthetic ligands behave as antagonists on the wild type but as agonists on the 5-HT4-RASSL. G-protein-coupled receptor receptor activated solely by synthetic ligands Dulbecco's modified Eagle's medium dialyzed fetal bovine serum phosphate-buffered saline transmembrane domain G protein-coupled receptors (GPCRs)1 are the most numerous and the most diverse type of receptors (1Bockaert J. Claeysen S. Becamel C. Pinloche S. Dumuis A. Int. Rev. Cytol. 2002; 212: 63-132Crossref PubMed Scopus (65) Google Scholar, 2Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1215) Google Scholar). They transduce messengers as different as odorants, nucleotides, nucleosides, peptides, lipids, or proteins. Eight families of GPCRs are classified according their sequences, their binding domains to ligands, and their ability to activate similar sets of G-proteins. The heterotrimeric (α, β, γ) G proteins are divided into four families based on the nature of their Gα subunits (Gαi, Gαs, Gαq, Gα12–13). Each one couples to a distinct class of receptors and signals through a specific pathway. A series of pathologies have been found to be related to the mutation of GPCRs. These changes lead either to a loss or a gain of function, such as blindness, diabetes insipidus, and hypo- or hyperthyroidism (3Spiegel A.M. Annu. Rev. Physiol. 1996; 58: 143-170Crossref PubMed Scopus (187) Google Scholar). Depressed GPCR signal transduction is related to numerous complex diseases. Heart failure and asthma are associated with a decrease in the Gs-signaling pathway, whereas an increase in Gi signaling is a potential cause of dilated cardiomyopathy). The concept that modified GPCRs could be used as tools to better understand GPCR-controlled signal transduction pathways in a given cell or organ or for gene therapy has recently been proposed (5Coward P. Chan S.D. Wada H.G. Humphries G.M. Conklin B.R. Anal. Biochem. 1999; 270: 242-248Crossref PubMed Scopus (202) Google Scholar, 6Scearce-Levie K. Coward P. Redfern C.H. Conklin B.R. Trends Pharmacol. Sci. 2001; 22: 414-420Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 7Scearce-Levie K. Coward P. Redfern C.H. Conklin B.R. Methods Enzymol. 2002; 343: 232-248Crossref PubMed Scopus (7) Google Scholar). Several types of modified GPCRs have been developed to prepare such tools. The first, engineered by Conklin and collaborators (8Coward P. Wada H.G. Falk M.S. Chan S.D. Meng F. Akil H. Conklin B.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 352-357Crossref PubMed Scopus (220) Google Scholar), were named RASSL for “receptor activated solely by synthetic ligands.” The idea is to engineer receptors that would be insensitive to their endogenous ligand(s) but can be fully activated by synthetic ligands. Among the GPCRs, only the κ opioid receptors have been modified so far by Conklin's group (8Coward P. Wada H.G. Falk M.S. Chan S.D. Meng F. Akil H. Conklin B.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 352-357Crossref PubMed Scopus (220) Google Scholar) to produce two opioid receptor-RASSLs, Ro1 and Ro2. Ro1 was constructed by substituting the second extracellular loop of the κ opioid receptor with the corresponding portion of the human δ opioid receptor. This substitution induced a lower affinity for the endogenous peptide ligands (including dynorphin) without significantly reducing the response to κ synthetic ligands, like spiradoline (8Coward P. Wada H.G. Falk M.S. Chan S.D. Meng F. Akil H. Conklin B.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 352-357Crossref PubMed Scopus (220) Google Scholar). The specificity of Ro1 for the synthetic ligands was further enhanced in Ro2 by substituting glutamine for Glu297 in Ro1. More recently, a second type of mutant GPCR the “therapeutic receptor-effector complex” (or “TREC”) was proposed to be a biotechnological tool to study GPCR signal transduction (9Small K.M. Brown K.M. Forbes S.L. Liggett S.B. J. Biol. Chem. 2001; 276: 31596-31601Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The β2-adrenergic receptor mutated in 19 positions and fused with Gαs was not activated by β-adrenergic agonists, but only by a non-biogenic amine agonist, L156870, although with relatively low potency. In this report, we describe a third example of RASSL type receptors: a mutant serotonin receptor, the 5-HT4 receptor. 5-HT4 receptors are Gs-coupled receptors expressed in the gastrointestinal tract, human and pig atria, urinary bladder, adrenal medulla, and central nervous system including limbic areas (olfactory tubercles, limbic system, basal ganglia) (10Bockaert J. Fagni L. Dumuis A. Baumgarten H.G. Göthert M. Handbook of Experimental Pharmacology: Serotoninergic Neurons and 5-HT Receptors in the CNS. Spinger-Verlag, Berlin1997: 439-465Google Scholar). Over the past 10 years, the pharmacology and structure of 5-HT4receptors have been extensively studied (10Bockaert J. Fagni L. Dumuis A. Baumgarten H.G. Göthert M. Handbook of Experimental Pharmacology: Serotoninergic Neurons and 5-HT Receptors in the CNS. Spinger-Verlag, Berlin1997: 439-465Google Scholar, 11Mialet J. Dahmoune Y. Lezoualc'h F. Berque-Bestel I. Eftekhari P. Hoebeke J. Sicsic S. Langlois M. Fischmeister R. Br. J. Pharmacol. 2000; 130: 527-538Crossref PubMed Scopus (53) Google Scholar, 12Joubert L. Claeysen S. Sebben M. Bessis A.S. Clark R.D. Martin R.S. Bockaert J. Dumuis A. J. Biol. Chem. 2002; 277: 25502-25511Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). One of the interesting characteristics of this receptor is its ability to be activated by a wide range of compounds from very different chemical classes (13Clark R.D. Eglen R.M. 5-HT4 Receptors in the Brain and Periphery. Springer-Verlag, Berlin1998: 1-48Crossref Google Scholar). Some are used in clinic to treat gastroparesis, dyspepsia gastro-esophageal reflux, or irritable bowel syndromes. The 5-HT4 agonists are related to tryptamines like 5-HT, to carbazimidamides (HTF-919 or Zelmac), benzamides (metoclopramide or PrimperanTM, cisapride or “PrepulsidTM,” benzoates (SL 10302), benzimidazolones (BIMU8), or aryl ketones (for reviews, see Refs. 10Bockaert J. Fagni L. Dumuis A. Baumgarten H.G. Göthert M. Handbook of Experimental Pharmacology: Serotoninergic Neurons and 5-HT Receptors in the CNS. Spinger-Verlag, Berlin1997: 439-465Google Scholar and 13Clark R.D. Eglen R.M. 5-HT4 Receptors in the Brain and Periphery. Springer-Verlag, Berlin1998: 1-48Crossref Google Scholar). The demonstration that tryptamines and benzamides have different pharmacophores (14Buchheit K.H. Gamse R. Giger R. Hoyer D. Klein F. Klöppner E. Pfannküche H.J. Mattes H. J. Med. Chem. 1995; 38: 2326-2330Crossref PubMed Scopus (71) Google Scholar) prompted us to generate mutant 5-HT4receptors with the aim of disrupting the 5-HT recognition site, keeping the recognition site for synthetic 5-HT4 agonists. The mutant was generated by exchanging the endogenous residue Asp100 to Ala in the m5-HT4(a)R cDNA sequence with the QuikChange site-directed mutagenesis kit (Stratagene). The sense primer used was: D100A, 5′-ACC TCT CTGGCT GTC CTA CTC ACC-3′. The cDNAs, subcloned into the pRK5 vector, were introduced into COS-7 cells by transfection. In summary, cells were trypsinized, centrifuged, and resuspended in EP Buffer (50 mm K2HPO4, 20 mm CH3CO2 K, 20 mm KOH, 26.7 mm MgSO4, pH 7.4) with 25–2000 ng of receptor cDNA. The total amount of cDNA was kept constant at 15 μg per transfection with pRK5 vector. After 15 min at room temperature, 300 μl of cell suspension (107 cells) were transferred to a 0.4-cm electroporation receptacle (Bio-Rad, Ivry sur Seine, France) and pulsed with a gene pulser apparatus (setting 1000 microfarads, 280 V). Cells were diluted in Dulbecco's modified Eagle's medium (DMEM; 106 cells/ml) containing 10% dialyzed and decomplemented fetal bovine serum (dFBS) and plated on 15-cm Falcon Petri dishes or into 12-well clusters at the desired density. Six hours after transfection, the surrounding cell medium was exchanged for DMEM without dFBS with 2 μCi of [3H]adenine/ml to label the ATP pool and incubated overnight (16 h). cAMP accumulation was measured, as described previously (15Dumuis A. Bouhelal R. Sebben M. Cory R. Bockaert J. Mol. Pharmacol. 1988; 34: 880-887PubMed Google Scholar). Membranes were prepared from transiently transfected cells plated on 15-cm dishes and grown in DMEM with 10% dFBS for 6 h, followed by incubation for 20 h in DMEM without dFBS. The cells were washed twice in PBS, scraped with a rubber policeman, harvested in PBS, and centrifuged at 4 °C (200 × g for 4 min). The pellet was resuspended in buffer containing 10 mmHEPES, pH 7.4, 5 mm EGTA, 1 mm EDTA, and 0.32m sucrose and homogenized 10 times with a glass-Teflon potter at 4 °C. The homogenate was centrifuged at 20,000 ×g for 20 min. The membrane pellet was resuspended in 50 mm HEPES, pH 7.4 (5 mg of protein in 1 ml of solution) and stored at −80 °C until use. Saturation experiments were performed using the specific 5-HT4 receptor radioligand [3H]GR 113808 at height concentrations ranging from 0.048 to 0.51 nm. The 5-HT4 receptor binding site density was estimated with [3H]GR 113808 at a saturating concentration (0.5 nm), as described previously (16Ansanay H. Sebben M. Bockaert J. Dumuis A. Eur. J. Pharmacol. 1996; 298: 165-174Crossref PubMed Scopus (39) Google Scholar). 5-HT (5 × 10−5m) or RS 100235 (10−7m), a 5-HT4 receptor antagonist, was used to determine nonspecific binding. Protein concentration in the samples was determined with the Bio-Rad protein assay. Competition and saturation experiments were analyzed by non-linear regression curves using the computer program LIGAND (17Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7759) Google Scholar). Saturation experiments were also analyzed according to Scatchard. IC50 values required to displace 50% of [3H]GR 113808 binding sites were converted toK D values, according to the equationK D = IC50/1 + S/K DS(18Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12132) Google Scholar), where S is [3H]GR 113808 concentration, andK DS is the equilibrium constant of [3H]GR 113808. Using Kaleidagraph software, the dose-response curves were fitted according to the following equation.Y=((ymax−ymin)/1+(x/EC50)nH))+yminEquation 1 where EC50 (or EC50inv) is the concentration of agonist (or inverse agonist) that evokes a half-maximal response, y max andy min correspond to the maximal and minimal responses, respectively, and nH is the Hill coefficient. Data were compared using the Stat-View Student program (Abacus Concepts, Berkeley, CA) with t tests. The following drugs were used: GR 113308 (1-[2-(methylsulfonylamino)ethyl]-4-piperidinyl 1-methyl-indole-3-carboxylate), HTF- 919 (5-methoxy-indole-3-carboxaldehyde 4-pentyl-iminosemicarbazone), BIMU8 (endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2-oxo-3-isopropyl-2,3-dihydro-1Hbenzimidazole-1-carboxamide), (S)-zacopride ((S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-4-amino-5-chloro-2-methoxy-benzamide monohydrochloride), cisapride (cis-N-[1-[3-(4-fluorophenoxy)propyl]-3-methoxy-4-piperidinyl]-4-amino-5-chloro-2-methoxy-benzamide, metoclopramide (N-(2-dimethylamino)ethyl)-4-amino-5-chloro-2-methoxybenzamide), renzapride ((±)-endo-N-(1-azabi-cyclo[3.3.1]non-4-yl)-4-amino-5-chloro-2-methoxybenzamide mo- nohydrochloride), SB 204070 ((1-butyl-4-piperidinyl)methyl 8-amino-7-chloro-1,4-benzodioxane-5-carboxylate), ML 10302 (2-(1-piperidinyl)ethyl-4-amino-5-chloro-2-methoxybenzoate), ML 10375 (2-(cis-3,5-dimethyl-1-piperidinyl)ethyl4-amino-5-chloro-2-methoxybenzoate), RS 67333 (1-(4-amino-5-chloro-2-methoxy-phenyl)-3-(1-butyl-4-piperidinyl)-1-propanone), RS 100350 (N-[2-[4-[3-(8-amino-7-chloro-1,4-benzodioxan-5-yl)-3-oxopropyl]-1-piperidinyl]ethyl]-4-methoxybenzenesulfonamide), RS 124523 (1-(8-amino-7-chloro-1,4-benzodioxan-5-yl)-3-[1-[3-(4-fluorophenyl)propyl]-4-piperidinyl]-1-propanone), RS 47431 (1-(4-amino-5-chloro-2-methoxy-phenyl)-3-[1-[3-(4-methoxyphenyl)propyl]-4-piperidinyl]-1-propanone), and RS 100235 (1-(8-amino- 7-chloro-1,4-benzodioxan-5-yl)-3-[1-[3-(3,4-dimethoxyphenyl)propyl]-4-piperidinyl]-1-propanone). First, we wanted to render the receptor unresponsive to 5-HT, but responsive to synthetic 5-HT4 agonists. Based on Strader's work on β-adrenergic receptors and on many other biogenic amine receptors (19Strader C. Fong T. Tota M. Underwood D. Dixon R. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (990) Google Scholar), the key interaction between 5-HT4receptors and 5-HT should obviously occur at the highly conserved Asp residue found in TM-III within the biogenic amine receptor family. In the 5-HT4 receptor this Asp is at position 100 in TM-III (Asp100(3.32)). This Asp residue is believed to interact with the positively charged nitrogen of the neurotransmitter protonated amine. Indeed, the mutation of Asp100(3.32) to alanine in the 5-HT4 receptor totally abolished the 5-HT stimulation of cAMP accumulation (11Mialet J. Dahmoune Y. Lezoualc'h F. Berque-Bestel I. Eftekhari P. Hoebeke J. Sicsic S. Langlois M. Fischmeister R. Br. J. Pharmacol. 2000; 130: 527-538Crossref PubMed Scopus (53) Google Scholar, 12Joubert L. Claeysen S. Sebben M. Bessis A.S. Clark R.D. Martin R.S. Bockaert J. Dumuis A. J. Biol. Chem. 2002; 277: 25502-25511Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), as well as the stimulation by other tryptamines (5-CT or 5-MeOT) or by a substituted indole carbazimidamide (HTF-919) (Fig. 1, A and B) (TableI). Compared with 5-HT, HTF-919 has a guanidine function instead of a protonated amine in the indole side chain (20Buchheit K. Gamse R. Giger R. Hoyer D. Klein F. Klöppner E. Pfannfucke H. Mattes H. J. Med. Chem. 1995; 38: 2331-2338Crossref PubMed Scopus (98) Google Scholar). Surprisingly, when we mutated other well conserved residues within the biogenic amine receptor family and likely to be involved in the 5-HT binding pocket (Ser197(5.43), Trp272(6.48), and Phe275(6.51)), none were able to totally suppress the 5-HT or HTF-919 responses. 5-HT has a 50–500-fold reduced affinity for F275A, W272A, and S197A, with no change in the maximal 5-HT stimulation (12Joubert L. Claeysen S. Sebben M. Bessis A.S. Clark R.D. Martin R.S. Bockaert J. Dumuis A. J. Biol. Chem. 2002; 277: 25502-25511Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar).Table I5-HT4 receptor drug efficiency on WT and D100(3.32)A mutant 5-HT4 receptorEfficiency was determined in cAMP accumulation experiments in COS-7 cells transiently expressing either 5-HT4(a) and mutant D100(3.32)A receptors at the same level of receptor density (about 2500 fmol/mg proteins). The results are expressed as percentages of 5-HT-induced cAMP accumulation via the 5-HT4 WT receptors and BIMU8-induced cAMP accumulation via the 5-HT4-R D100(3.32)A mutant receptors. Data are the means ± S.E. values of three experiments performed in triplicate (see “Experimental Procedures”). The chemical structures of some of the 5-HT4receptor drugs tested in this study are shown. Open table in a new tab Efficiency was determined in cAMP accumulation experiments in COS-7 cells transiently expressing either 5-HT4(a) and mutant D100(3.32)A receptors at the same level of receptor density (about 2500 fmol/mg proteins). The results are expressed as percentages of 5-HT-induced cAMP accumulation via the 5-HT4 WT receptors and BIMU8-induced cAMP accumulation via the 5-HT4-R D100(3.32)A mutant receptors. Data are the means ± S.E. values of three experiments performed in triplicate (see “Experimental Procedures”). The chemical structures of some of the 5-HT4receptor drugs tested in this study are shown. Interestingly, and in contrast to other biogenic amine receptors (9Small K.M. Brown K.M. Forbes S.L. Liggett S.B. J. Biol. Chem. 2001; 276: 31596-31601Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), the mutation of the conserved Asp100(3.32) only moderately affected the specific 5-HT4R radioligand, [3H]GR 113808 (K D = 0.17 ± 0.08 nm and 0.38 ± 0.09 nm at WT and D100(3.32)A receptors, respectively) (Fig. 1 D). Using [3H]GR 113808 binding, we verified that 5-HT was unable to bind the 5-HT4 D100(3.32)A receptor (Fig 1 C). The binding of [3H]GR 113808 was the first indication that ligands, which include the basic nitrogen of the aromatic ring side chain, in a structured ring, associated with an increase in the distance between this basic nitrogen and the main aromatic ring of the compound, suppressed the requirement of the Asp100(3.32) carboxylic group for ligand binding. This was confirmed when we screened most of the non-tryptamine 5-HT4 receptor agonists for their ability to activate the 5-HT4 D100(3.32)A receptor. BIMU8, a specific 5-HT4 agonist structurally very different from 5-HT, belonging to the azabicycloalyl benzymidazolone class (21Dumuis A. Sebben M. Monferini E. Nicola M. Ladinsky H. Bockaert J. Naunyn-Schmiedeberg's Arch. Pharmacol. 1991; 343: 245-251Crossref PubMed Scopus (53) Google Scholar), was found to be a potent agonist. As shown in Fig. 2, this compound remained fully active and even showed higher efficacy and potency on the D100A mutant than on the WT (EC50 values for cAMP stimulation were 4 ± 1.5 and 1 ± 0.5 nm for WT and D100A, respectively) (Fig. 2 A). The affinity of BIMU8 for the 5-HT4receptor was also slightly better on the D100A mutant than on the WT.K D values for BIMU8, measured by competition with the [3H]GR 113808 radioligand, were 30 ± 11 nm and 6.5 ± 3 nm for WT and D100A, respectively (Fig. 2 B). Similarly, the benzamides bearing the 2-methoxy-4-amino-5-chloro substitution (renzapride, S-zacopride, or cisapride) were equi-effective on the WT and the D100(3.32)A mutant (Table I) with nanomolar affinity. A slight decrease in their potency on the mutant D100A receptor was observed. These data suggest that the D100(3.32)A mutant could still be activated as long as the agonist could bind the receptor. Furthermore, this mutation had no effect on the receptor expression level. Two drugs were antagonists on WT and agonists at the D100(3.32)A mutant. Benzoate derivatives bearing the 2-methoxy-4-amino-5-chloro-substitution (ML 10302) (22Langlois M. Zhang L. Yang D. Brémont B. Shen S. Manara L. Croci T. Bioorg. Med. Chem. Lett. 1994; 4: 1433-1436Crossref Scopus (36) Google Scholar) and the related aryl ketones (RS 67333, RS 124523, RS 100350, RS 47431) (13Clark R.D. Eglen R.M. 5-HT4 Receptors in the Brain and Periphery. Springer-Verlag, Berlin1998: 1-48Crossref Google Scholar) were partial agonists on 5-HT4 WT receptors. As shown in TableI, they were more efficacious on 5-HT4 D100(3.32)A receptors than on WT. Another benzoate derivative, reported to be either a highly specific 5-HT4 antagonist (ML 10375) (23Yang D. Soulier J.L. Sicsic S. Mathe-Allainmat M. Bremont B. Croci T. Cardamone R. Aureggi G. Langlois M. J. Med. Chem. 1997; 40: 608-621Crossref PubMed Scopus (66) Google Scholar) or an inverse agonist on the 5-HT4 receptor (24Blondel O. Gastineau M. Langlois M. Fischmeister R. Br. J. Pharmacol. 1998; 125: 595-597Crossref PubMed Scopus (35) Google Scholar), was indeed an antagonist of WT 5-HT4 receptors (Fig. 3 C), which became a full agonist on the 5-HT4 D100(3.32)A mutant (EC50 = 1 ± 0.6 nm) (Fig. 3, A and C). This encouraged us to screen the putative agonist properties of a series of 5-HT4 antagonists. We found that the known GR 113808 antagonist (Fig. 3 C) used for 5-HT4receptor studies (25Grossman C.J. Kilpatrick G.J. Bunce K.T. Br. J. Pharmacol. 1993; 109: 618-624Crossref PubMed Scopus (284) Google Scholar) was a highly potent (EC50 = 0.17 ± 0.04 nm) and efficacious agonist on D100(3.32)A receptor (Fig. 3 B). Compared with the previously described β2-adrenergic Gs-coupled RASSL (9Small K.M. Brown K.M. Forbes S.L. Liggett S.B. J. Biol. Chem. 2001; 276: 31596-31601Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), the 5-HT4 RASSL has the advantage of being stimulated by synthetic compounds of much higher affinity (micromolar for β2-adrenergic RASSL and nanomolar for 5-HT4 RASSL). The structure-activity relationships and the structural analyses of the 5-HT4 receptor ligands used in this study are consistent with a recent report on comparative receptor mapping of 5-HT4 binding sites (26Lopez-Rodriguez M.L. Murcia M. Benhamu B. Viso A. Campillo M. Pardo L. Bioorg. Med. Chem. Lett. 2001; 11: 2807-2811Crossref PubMed Scopus (20) Google Scholar). The authors proposed structural insights to assist the design of selective 5-HT4 receptor ligands. The structural features that define the 5-HT4ligands are an aromatic moiety, a coplanar carbonyl, carboxyl or ketone function, and a basic nitrogen atom. The substitute of the basic nitrogen must be voluminous (as in all the active drugs acting on the 5-HT4 RASSL) to interact in an hydrophobic pocket of the receptor. The size of the substitute improves selectivity and potency. One of our hypotheses is that the basic nitrogen of these drugs interacts within the hydrophobic pocket, with a negative charge, but not with Asp(3.32). In contrast, this Asp is necessary for the binding of the protonated primary amine of 5-HT derivatives. The affinity of the voluminous substituted ligands for the 5-HT4 RASSL (structures in Table I) would depend of the interactions between their hydrophobic rings and the hydrophobic pocket, defined by highly conserved aromatic residues in TMVI and VII (Trp(6.48), Phe(6.51), and Tyr(7.43)) (11Mialet J. Dahmoune Y. Lezoualc'h F. Berque-Bestel I. Eftekhari P. Hoebeke J. Sicsic S. Langlois M. Fischmeister R. Br. J. Pharmacol. 2000; 130: 527-538Crossref PubMed Scopus (53) Google Scholar, 27Roth B.L. Shoham M. Choudhary M.S. Khan N. Mol. Pharmacol. 1997; 52: 259-266Crossref PubMed Scopus (129) Google Scholar, 28Lopez-Rodriguez M.L. Benhamu B. Viso A. Morcillo M.J. Murcia M. Orenzanz L. Alfaro M.J. Martin M.I. Bioorg. Med. Chem. 1999; 7: 2271-2281Crossref PubMed Scopus (36) Google Scholar). The above results indicate that the D100(3.32)A Gs-coupled 5-HT4 receptor described here is the first RASSL related to serotonin receptors and that it has unique properties not found in two previously described RASSL receptors (5Coward P. Chan S.D. Wada H.G. Humphries G.M. Conklin B.R. Anal. Biochem. 1999; 270: 242-248Crossref PubMed Scopus (202) Google Scholar, 7Scearce-Levie K. Coward P. Redfern C.H. Conklin B.R. Methods Enzymol. 2002; 343: 232-248Crossref PubMed Scopus (7) Google Scholar). RASSL is generated by a single mutation and is completely insensitive to its endogenous agonist: 5-HT. RASSL can be stimulated by numerous synthetic agonists from different chemical classes. Many of these synthetic ligands have a nanomolar affinity for the 5-HT4 RASSL, and some of them can be administered orally such as cisapride or metoclopramide. Cisapride has been used for years to treat dyspepsia and gastro-esophageal reflux and has only been removed from the market because of one second effect (arrhythmia probably caused by action on K+ channels) (29Kii Y. Ito T. J. Cardiovasc. Pharmacol. 1997; 29: 670-675Crossref PubMed Scopus (43) Google Scholar, 30Kaumann A.J. Lynham J.A. Brown A.M. Naunyn-Schmiedeberg's Arch. Pharmacol. 1996; 353: 592-595Crossref PubMed Google Scholar). However, other benzamides are being developed and will certainly be used in humans. Interestingly, RS 67333 was found to be another potent agonist of 5-HT4 RASSL. This compound possesses a great ability to cross the blood-brain barrier. Antagonist compounds (ML 10375 and GR 113808), which are highly specific on the native receptor, exhibit agonist properties on the 5-HT4 RASSL. GR 113808 is also able to cross the blood-brain barrier. ML 10375 and GR 113808 will be particularly interesting for in vivo stimulation of 5-HT4RASSL. Indeed, with these compounds, native 5-HT4 receptors can be kept silent, while generating a Gs signal by activating 5-HT4 RASSL, expressed in a given tissue at a given developmental time, by bioengineering in animals and possibly in humans.