Research reportMolecular biology of 5-HT receptors
Introduction
Serotonin (5-hydroxytryptamine; 5-HT) as a neurotransmitter acts via membrane receptors in the central nervous (CNS) and the peripheral nervous system (PNS), as well as in non-neuronal tissues (e.g. blood, gastro intestinal, endocrine, sensory and cardiovascular systems, to name a few). 5-HT is one of the oldest neurotransmitters/hormones in evolution, and its receptors are estimated to have appeared 700–800 Mio years in single cell eukaryotes such as paramecia; 5-HT receptors are found in such diverse species from planaria, c. Elegans, and drosophila to man are, and are rather well conserved. This may explain why 5-HT interacts with such a diversity of receptors of the G-protein-coupled family and the ligand-gated family, similarly to acetylcholine, GABA or glutamate, but with more receptor subtypes and a larger diversity at play. They may actually have been amongst the first rhodopsin-like receptors reacting to a chemical. The major classes of 5-HT receptors must have diverged about 750 millions years ago, long before cholinergic, adrenergic or dopaminergic receptors, although the GPCR family may date from >1 billion years. Serotonin was first described as enteramine, which was isolated from the gut in the 1930s by Ersparmer and colleagues and shown to cause contraction of the uterus. 5-HT was rediscovered in the 1940s by Irvin Page's group in the circulation and called serotonin, based on its vasoconstrictor features (although it also relaxes blood vessels); eventually Maurice Rapport purified, crystallized and characterized the molecule from vast amounts of blood. Rapport found that enteramine and serotonin covered the same entity, namely 5-hydroxytryptamine, which indeed contained an indole as already suggested by Ersparmer and eventually the two groups came to the same conclusion. The availability of synthetic 5-HT was the real start of the 5-HT saga in pharmacological terms.
The subdivision of 5-HT receptors started in the 1950s by Gaddum and colleagues, when they realised that in the guinea pig ileum, the effects of 5-HT could be blocked in part by morphine (M), and in part by dibenzyline (D). Gaddum and Picarelli proposed two receptor classes, 5-HT M and 5-HT D (1957). Although non-selective tools were used, the concept proved to be correct. In 1976, when after many painful attempts at monoamine receptor binding, the first radioligand-binding studies succeeded using [3H]5-HT and [3H]LSD, Fillion and colleagues suggested the existence of 5-HT receptors in brain labeled with [3H]5-HT and [3H]LSD (1976, 1977, 1978, 1979); however these papers did not get the deserved attention of the community. Then in 1979, Peroutka and Snyder described two classes of brain 5-HT receptors, using [3H]-5-HT, [3H]-spiperone (a dopaminergic ligand), and [3H]-LSD called 5-HT1 ([3H]-5-HT binding) and 5-HT2 ([3H]-spiperone), with [3H]-LSD labeling both classes. Interestingly, Gaddum's M receptor was still distinct from the 5-HT1 and 5-HT2 receptors in both function and distribution, whereas the D receptor resembled pharmacologically the 5-HT2 binding site; it was also assumed for quite some time that the 5-HT M receptor was purely peripheral, as amply documented in functional studies, and most of the 5-HT stems from chromaffin cells in the gut, whereas in the brain much of the serotonin comes from the raphé nuclei. Thus, Phillip Bradley convened a party in charge of unifying the 5-HT receptor concept and nomenclature. Bradley et al. [1] proposed the existence three families of 5-HT receptors, named 5-HT1-like (there was already suggestions for diversity of this group from radioligand binding and autoradiographic studies), 5-HT2 and 5-HT3, the latter corresponding to the M receptor. The proposal was based primarily on functional criteria, since radioligand binding was still not convincing to many colleagues, second messenger studies were less popular than today, and no GPCR had been cloned at the time that party congregated from 1984 on; nevertheless, the Bradley nomenclature represented a useful classification framework. However, with the increasing use of radioligand binding in membranes and cells, autoradiography in brain slices and second messenger studies in cells and tissue, subtypes of 5-HT1 receptor binding sites were further described (5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, 5-ht1E). It became rapidly evident that the 5-HT1C receptor found in the choroid plexus (although labeled with high affinity by [3H]-5-HT), is closer the 5-HT2 receptor family, due to similar pharmacological profiles and 2nd messenger features (stimulation of inositol phosphate production and calcium signalling), and this suggested the existence of 5-HT2 receptor subtypes as well. Further, another 5-HT receptor which had been identified in the mid 1980's in the gastrointestinal tract, heart and brain, was termed 5-HT4 by Saxena and colleagues (see [2]), but this proposal was initially rejected. Fortunately, in 1986–1988, the molecular biology era started with the cloning of the 5-HT1A receptor (interestingly, G21 as the 5-HT1A receptor was called when it was still an orphan, allowed the cloning of further beta adrenoceptors by homology, see [3], [4]). Rapidly, most known but also some unsuspected 5-HT receptors were cloned. This work led to the identification of a number of ‘new’ receptors, devoid of immediate physiological counterparts. Tentatively termed 5-ht1E, 5-HT1F, 5-HT2F, 5-HT5A, 5-HT5B, 5-HT6, and 5-HT7, they required integration into an acceptable classification scheme. As is known now, all 5-HT receptors that belong to the GPCR are part of the type A family of GPCRs and show still significant sequence homology with rhodopsin, but also adrenergic and dopaminergic receptors, and contain the famous DRY motif in the third transmembrane spanning region. Based on these new findings and yet a constantly evolving field, the Serotonin Club Receptor Nomenclature Committee proposed a new nomenclature system based on operational, structural and transductional information ([5], see Table 1). These principles were subsequently applied to a number of receptor families by the newly created Receptor Nomenclature Committee of the International Union of Pharmacology (NC-IUPHAR), keeping in mind that some historical features will have to be acknowledged at least transiently (for instance the reclassification of opiate receptors met quite some resistance and we are left with the former mu, delta and kappa opiate receptor classes). The current classification ([6]; Hoyer et al. [214]) is flexible and intended to be adapted, as information from both recombinant and native systems becomes available; however it favours an alignment of nomenclature with the human genome to avoid species differences (see [7], [8]). Seven families of 5-HT receptors form the basis of the classification, based on pharmacology, transduction and structure, although one could argue that structure must be predominant as it governs function and ultimately pharmacological signature. One orphan receptor, called 5-HT1P by Gershon and co-workers, present in the gut [9], is not structurally characterized, and it remains to be seen whether this receptor is a homomer (new) or possibly a heterodimer composed of already known receptors. 5-HT3 receptors are ligand-gated ion channels, thus the 5-HT receptor family is showing similar diversity as do the acetylcholine or glutamate receptors families, which act through both metabotropic receptors and some fast acting ligand-gated channels. The 5-HT system has long been known to regulate emotions, behavioral control and cognition in a very complex manner, as has become evident from a great number of animal and human studies (see [10]); and that complexity may not be too surprising given the number of players involved in the 5-HT system which includes a multiplicity of receptors, transporters and metabolizing enzymes.
Section snippets
5-HT1 receptors, a family of receptors coupled to Gi/o
Class A GPCRs can be subdivided depending on their coupling to second messengers via the G-proteins and 5-HT1 receptors are mostly linked to Gi/o, which are pertussis toxin sensitive and couple negatively to adenylate cyclase; in cells, this may lead to membrane depolarization and inhibition of firing. The 5-HT1 receptor class is composed of five receptors (5-HT1A, 5-HT1B, 5-HT1D, 5-ht1e and 5-HT1F) which, in humans, share 40–63% overall sequence identity and couple somewhat preferentially to G
5-HT2 receptors, a family of receptors coupled to Gq/11
There are three types of 5-HT2 receptors. 5-HT2A, 5-HT2B and 5-HT2C receptors Exhibit 46–50% overall sequence identity and couple preferentially to Gq/11 to increase inositol phosphates and cytosolic [Ca2+] and in agreement with their long known role in muscle contraction and stimulation in the brain. 5-HT2 receptors may also couple to G12/13 which are known to mediate long term structural changes in cells. The 5-HT2A receptor refers to the classical D receptor initially described by Gaddum and
5-HT3 receptors, a ligand-gated ion channel receptor of the cys-loop channel family
5-HT3 receptors equate with the M receptor of Gaddum and Picarelli [89]; they belong to the ligand-gated ion channel receptor superfamily, similarly to the nicotinic acetylcholine, glycine or GABA-A receptors and share electrophysiological and structural patterns with the Cys-loop transmitter-gated superfamily of ligand-gated ion channels [140]. There are antagonist ligands that share affinity at 5-HT3 and nicotine receptors, such as tropisetron (ICS-205930). 5-HT3 receptors are located on
5-HT receptors that preferentially couple to Gs
5-HT4, 5-HT6 and 5-HT7 receptors all couple preferentially to Gs and promote cAMP formation, by activation of various adenylate cyclases. In turn, cAMP as an intracellular messenger interacts with various targets, the phosphorylating enzyme protein kinase A (PKA), but also cyclic nucleotide-gated ion channels, leading to the modulation of calcium ion flux and membrane excitability, other cellular processes. PKA phosphorylates cAMP-responsive transcription factors, such as the cAMP response
Conclusion
The 5-HT receptor family is complex, and one may ask as does Bryan Roth et al. [205] whether this is useless diversity (i.e. too much redundancy) or an embarrassment of the riches (i.e. many potential targets to choose from to affect normal or pathological function); molecular biology has largely confirmed, but also significantly enlarged the suspected diversity and stopped discussions about too much complexity or redundancy of 5-HT receptors. It still remains to be seen which functions some of
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