Molecular biology of serotonin (5-HT) receptors (original) (raw)
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Molecular Biology of Serotonin Receptors - Structure and Function at the Molecular Level
Current Topics in Medicinal Chemistry, 2002
5-hydroxytryptamine (5-HT; serotonin) is a neurotransmitter essential for a large number of physiological processes including the regulation of vascular and non-vascular smooth muscle contraction, modulation of platelet aggregation, and the regulation of appetite, mood, anxiety, wakefulness and perception. To mediate this astonishing array of functions, no fewer than 15 separate receptors have evolved, of which all but two (5-HT 3A and 5-HT 3B) are G-protein coupled receptors. This review will summarize our current understanding of the structure and function of the G-protein coupled 5-HT receptors. In particular, a systematic review of the available mutagenesis studies of 5-HT receptors will be presented. This information will be synthesized to provide a working model of agonist and antagonist actions at a prototypic 5-HT receptor-the 5-HT 2A receptor. Finally, examples will be given to demonstrate that a detailed knowledge of the predicted structure of one receptor can be useful for structure-based drug design.
Journal of Biological Chemistry, 2005
Although dimerization appears to be a common property of G-protein-coupled receptors (GPCRs), it remains unclear whether a GPCR dimer binds one or two molecules of ligand and whether ligand binding results in activation of one or two G-proteins when measured using functional assays in intact living cells. Previously, we demonstrated that serotonin 5-hydroxytryptamine2C (5-HT 2C) receptors form homodimers (Herrick-Davis, K., Grinde, E., and Mazurkiewicz, J. (2004) Biochemistry 43, 13963-13971). In the present study, an inactive 5-HT 2C receptor was created and coexpressed with wild-type 5-HT 2C receptors to determine whether dimerization regulates receptor function and to determine the ligand/dimer/G-protein stoichiometry in living cells. Mutagenesis of Ser 138 to Arg (S138R) produced a 5-HT 2C receptor incapable of binding ligand or stimulating inositol phosphate (IP) signaling. Confocal fluorescence imaging revealed plasma membrane expression of yellow fluorescent protein-tagged S138R receptors. Expression of wild-type 5-HT 2C receptors in an S138R-expressing stable cell line had no effect on ligand binding to wild-type 5-HT 2C receptors, but inhibited basal and 5-HT-stimulated IP signaling as well as constitutive and 5-HT-stimulated endocytosis of wild-type 5-HT 2C receptors. M1 muscarinic receptor activation of IP production was normal in the S138R-expressing cells. Heterodimerization of S138R with wild-type 5-HT 2C receptors was visualized in living cells using confocal fluorescence resonance energy transfer (FRET). FRET was dependent on the donor/acceptor ratio and independent of the receptor expression level. Therefore, inactive 5-HT 2C receptors inhibit wild-type 5-HT 2C receptor function by forming nonfunctional heterodimers expressed on the plasma membrane. These results are consistent with a model in which one GPCR dimer binds two molecules of ligand and one G-protein and indicate that dimerization is essential for 5-HT receptor function. G-protein-coupled receptors (GPCRs) 2 represent one of the largest families of signaling proteins in the human genome and are targets for a wide variety of therapeutic agents. Recent studies investigating GPCR structure and function indicate that they form dimeric or oligomeric * This work was supported by National Institutes of Health Grants MH057019 (to K. H.-D.
Journal of Biological Chemistry, 1996
Like other amine neurotransmitters that activate Gprotein-coupled receptors, 5-hydroxytryptamine (5-HT) binds to the 5-HT 2A receptor through the interaction of its cationic primary amino group with the conserved Asp 3.32(155) in transmembrane helix 3. Computational experiments with a 5-HT 2A receptor model suggest that the same functional group of 5-hydroxytryptamine also forms a hydrogen bond with the side chain of Ser 3.36(159) , which is adjacent in space to Asp 3.32(155). However, other 5-HT 2A receptor ligands like lysergic acid diethylamide (LSD), in which the amine nitrogen is embedded in a heterocycle, or N,N-dimethyl 5-HT, in which the side chain is a tertiary amine, are found in the computational simulations to interact with the aspartate but not with the serine, due mainly to steric hindrance. The predicted difference in the interaction of various ligands in the same receptor binding pocket was tested with site-directed mutagenesis of Ser 3.36(159) 3 Ala and Ser 3.36(159) 3 Cys. The alanine substitution led to an 18-fold reduction in 5-HT affinity and the cysteine substitution to an intermediate 5-fold decrease. LSD affinity, in contrast, was unaffected by either mutation. N,N-Dimethyl 5-HT affinity was unaffected by the cysteine mutation and had a comparatively small 3-fold decrease in affinity for the alanine mutant. These findings identify a mode of ligand-receptor complexation that involves two receptor side chains interacting with the same functional group of specific serotonergic ligands. This interaction serves to orient the ligands in the binding pocket and may influence the degree of receptor activation.
Journal of Biological Chemistry
Signaling through serotonin 6-HT1A receptors involves multiple pathways. We have investigated the functional coupling of the human 5-HT1A receptor to different G proteins using an in vitro reconstitution system based on the expression of recombinant receptor (r6-HTlA) and Ga-subunits (rG,) in Escherichia coli. The r5-HT1A receptor was expressed by insertion in a vector allowing its active expression in E. coli inner membranes. Binding of the selective agonist t3H] +8-hydroxy-(2-N-dipropylamine)tetralin (I3H]8-0H-DPAT) to intact bacteria or E. coli membranes was saturable with a KO of -8 nM and an average of 120 sitesbacterium. Binding properties of several serotoninergic ligands to r5-HT1A receptors were comparable with those measured in mammalian cells.
Comparative receptor mapping of serotoninergic 5-HT3 and 5-HT4 binding sites
Jcamd, 1998
The clinical use of currently available drugs acting at the 5-HT 4 receptor has been hampered by their lack of selectivity over 5-HT 3 binding sites. For this reason, there is considerable interest in the medicinal chemistry of these serotonin receptor subtypes, and significant effort has been made towards the discovery of potent and selective ligands. Computer-aided conformational analysis was used to characterize serotoninergic 5-HT 3 and 5-HT 4 receptor recognition. On the basis of the generally accepted model of the 5-HT 3 antagonist pharmacophore, we have performed a receptor mapping of this receptor binding site, following the active analog approach (AAA) defined by Marshall. The receptor excluded volume was calculated as the union of the van der Waals density maps of nine active ligands (pK i ≥ 8.9), superimposed in pharmacophoric conformations. Six inactive analogs (pK i < 7.0) were subsequently used to define the essential volume, which in its turn can be used to define the regions of steric intolerance of the 5-HT 3 receptor. Five active ligands (pK i ≥ 9.3) at 5-HT 4 receptors were used to construct an antagonist pharmacophore for this receptor, and to determine its excluded volume by superimposition of pharmacophoric conformations. The volume defined by the superimposition of five inactive 5-HT 4 receptor analogs that possess the pharmacophoric elements (pK i ≤ 6.6) did not exceed the excluded volume calculated for this receptor. In this case, the inactivity may be due to the lack of positive interaction of the amino moiety with a hypothetical hydrophobic pocket, which would interact with the voluminous substituents of the basic nitrogen of active ligands. The difference between the excluded volumes of both receptors has confirmed that the main difference is indeed in the basic moiety. Thus, the 5-HT 3 receptor can only accommodate small substituents in the position of the nitrogen atom, whereas the 5-HT 4 receptor requires more voluminous groups. Also, the basic nitrogen is located at ca. 8.0 Å from the aromatic moiety in the 5-HT 4 antagonist pharmacophore, whereas this distance is ca. 7.5 Å in the 5-HT 3 antagonist model. The comparative mapping of both serotoninergic receptors has allowed us to confirm the three-component pharmacophore accepted for the 5-HT 3 receptor, as well as to propose a steric model for the 5-HT 4 receptor binding site. This study offers structural insights to aid the design of new selective ligands, and the resulting models have received some support from the synthesis of two new active and selective ligands: 24 (K i (5-HT 3 ) = 3.7 nM; K i (5-HT 4 ) > 1000 nM) and 25 (K i (5-HT 4 ) = 13.7 nM; K i (5-HT 3 ) > 10 000 nM).
GENERAL BACKGROUND OF 5 -HYDROXYTRYPTAMINE AND RECEPTORS
5-Hydroxytryptamine (5-HT, serotonin) is a common biogenic amine found in both vertebrates and invertebrates as well as in plants . The precursor to 5-HT, tryptophan, is likey important in the early evolution of life and perhaps the early presence of tryptophan is a reason for 5-HT to be potentially the first neurotransmitter noted with the development of a nervous system . 5-HT acts as both a neurotransmitter and neurohormone and as a potent modulator of neurons and various tissues in many animal species . Generally 5-HT actions are elicited by transmembrane G protein coupled receptors (GPCRs), which then activate or inhibit different intracellular second messenger cascades. 5-HT receptors from some organisms have been classified based on sequence or pharmacology ; for example in the vertebrates, 7 families (5-HT 1-7 ), 14 subtypes have been identified, whereas in Drosophila four 5-HT receptors named 5-HT 1Adro 5-HT 1Bdro 5-HT 2dro 5-HT 7dro [5-10] have been classified. 5-HT receptors appear to be present on invertebrate presynaptic nerve terminals and on muscle membranes; receptors of a cricket (Gryllus domestica) mandibular muscle have a similar pharmacological profile as a 5-HT 2 -like receptor subtype . Profiling the 5-HT receptor subtypes directly on skeletal muscle within invertebrates is an area of research that is lacking. The 5-HT 4 and 5-HT 7 receptors are shown to have alternate splice variants which increase the number of receptor subtypes and may
Proceedings of the National Academy of Sciences, 1990
Serotonin exerts its diverse physiological effects by interacting with multiple distinct receptor subtypes. We have isolated a rat brain 5HT2 serotonin receptor cDNA by virtue of its homology with the 5HT1c receptor. The 5HT2 receptor is a member of the family of receptors that are linked to guanine nucleotide-binding proteins and are predicted to span the lipid bilayer seven times. Overall sequence identity between the 5HT2 and 5HT1c receptors is 49%, but identity within the transmembrane domains is 80%. Expression of both the 5HT2 and 5HT1c receptors in transfected mouse fibroblasts activates phospholipase C signaling pathways and promotes cellular transformation. However, RNA blotting shows that these two receptor subtypes are differentially expressed in the central nervous system. In this manner, structurally and functionally homologous receptor subtypes may elicit distinct physiologic actions.
Journal of Biological Chemistry, 2002
Bacteriorhodopsin and rhodopsin crystal structures were used as templates to build structural models of the mouse and human serotonin (5-HT)-2B receptors (5-HT 2B Rs). Serotonin was docked to the receptors, and the amino acids predicted to participate to its binding were subjected to mutagenesis. 5-HT binding affinity and 5-HT-induced inositol triphosphate production were measured in LMTK ؊ cells transfected with either wildtype or mutated receptor genes. According to these measurements, the bacteriorhodopsin-based models of the 5-HT 2B Rs appear more confident than the rhodopsin-based ones. Residues belonging to the transmembrane domains 3 and 6, i.e. Asp 3.32 , Ser 3.36 , Phe 6.52 , and Asn 6.55 , make direct contacts with 5-HT. In addition, Trp 3.28 , Phe 3.35 , Phe 6.52 , and Phe 7.38 form an aromatic box surrounding 5-HT. The specificity of human and mouse 5-HT 2B Rs may be reflected by different rearrangements of the aromatic network upon 5-HT binding. Two amino acids close to Pro 5.50 in the human transmembrane domain 5 sequence were permuted to introduce a "mouse-like" sequence. This change was enough to confer the human 5-HT 2B R properties similar to those of the mouse. Taken together, the computed models and the site-directed mutagenesis experiments give a structural explanation to (i) the different 5-HT pK D values measured with the human and mouse 5-HT 2B Rs (7.9 and 5.8, respectively) and (ii) the specificity of 5-HT binding to 5-HT 2B Rs as compared with other serotonergic Gprotein coupled receptors.