The human 5-ht5A receptor couples to Gi/Go proteins and inhibits adenylate cyclase in HEK 293 cells
Introduction
The neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) is involved in the control of diverse physiological processes including sleep, sexual behaviour, food intake, locomotion and mood. Depression, schizophrenia and migraine are among the pathological conditions associated with a dysfunction of 5-HT transmission.
At least thirteen different 5-HT receptors have been identified to date. One, the 5-HT3 receptor, belongs to the superfamily of ligand-gated ion channels; the others are seven-transmembrane-domain receptors that couple to heterotrimeric guanine nucleotide-binding proteins (G proteins). The G protein-coupled 5-HT receptors are classified into six subfamilies (5-HT1, 5-HT2, 5-HT4, 5-ht5, 5-ht6 and 5-HT7) (for review, Saudou and Hen, 1994; Hoyer and Martin, 1997).
The two members of the 5-ht5 receptor subfamily, 5-ht5A and 5-ht5B, were first identified in mice (Plassat et al., 1992; Matthes et al., 1993) and subsequently in rats (Erlander et al., 1993; Wisden et al., 1993). A cDNA encoding the 5-ht5A receptor has been cloned from human tissue, while the 5-ht5B receptor does not seem to be functionally expressed in humans (Rees et al., 1994; Grailhe et al., 1995). Pharmacologically, 5-ht5 receptors resemble the 5-HT1 subfamily of receptors, displaying high affinities for the agonist 5-CT and ergot derivatives, such as LSD and ergotamine. However, for various reasons 5-ht5 receptors may represent a distinct subfamily. In contrast to 5-HT1 receptor genes, 5-ht5 genes contain an intron in the region encoding the third intracellular loop (Plassat et al., 1992). Furthermore, 5-ht5 receptors exhibit a rather low affinity for 5-HT and the percentage of amino acid sequence homology in the transmembrane domains to other 5-HT receptors is less than 50% (Humphrey, 1997).
The physiological function of 5-ht5 receptors is still unclear. At present, no selective ligands are available. Possible functions have been postulated based on localisation studies, using Northern blot analysis, reverse transcriptase-polymerase chain reaction, in situ hybridisation and immunohistochemistry. For example, the limbic distribution (dendate gyrus and CA1/CA3 regions of the hippocampus, amygdala, entorhinal cortex) of 5-ht5A mRNA in the mouse, rat and human brain suggests a possible role in learning, memory and emotional behaviour (Plassat et al., 1992; Erlander et al., 1993; Pasqualetti et al., 1998), while the presence of 5-ht5A mRNA in the cerebellum may implicate a role in motor co-ordination and control (Plassat et al., 1992; Carson et al., 1996; Pasqualetti et al., 1998). 5-ht5A mRNA was also detected in the habenula (Plassat et al., 1992; Matthes et al., 1993), suggesting a possible role for 5-ht5A receptors in the acquisition of adaptive behaviour under stressful situations (Branchek and Zgombick, 1997). Recently, knockout mice lacking the 5-ht5A receptor were generated. These mutant mice were shown to have enhanced exploratory activity in a novel environment, suggesting that 5-ht5A receptors might modulate exploratory behaviour (Grailhe et al., 1997).
The mouse, rat and human 5-ht5 receptors have already been expressed in various cell systems, but most of these reported studies failed to demonstrate effects on signal transduction systems, such as adenylate cyclase or phospholipase C (Plassat et al., 1992; Matthes et al., 1993; Erlander et al., 1993; Wisden et al., 1993). Although no second messenger coupling could be detected for the mouse 5-ht5A receptor, a GTP-sensitive binding site was described when the receptor was expressed at low density in NIH 3T3 cells (Plassat et al., 1992). Furthermore, high-affinity agonist radioligand ([]5-CT) binding to the rat 5-ht5B receptor expressed in COS-1 cells could be shifted by the addition of Gpp(NH)p (Wisden et al., 1993). Recently, however, the rat 5-ht5A receptor expressed in C6 glioma cells was reported to be negatively coupled to adenylate cyclase activity (Carson et al., 1996). In addition, during the preparation of this manuscript, Hurley et al. (1998)reported agonist-induced stimulation of []GTPγS binding and inhibition of adenylate cyclase activity for the human 5-ht5A receptor, expressed in HEK 293 cells.
In the present study, we have extensively investigated the binding properties, G protein coupling and activation of second messenger responses of the human (h) 5-ht5A receptor expressed in stably transfected human embryonic kidney (HEK) 293 (h5-ht5A-HEK 293) cells. We have determined the effect of receptor-G protein uncoupling on high-affinity agonist ([]5-CT) binding, by the addition of Gpp(NH)p and pre-treatment with pertussis toxin. We have investigated h5-ht5A receptor-mediated activation of G proteins, using agonist-stimulated []GTPγS binding and we have explored 5-HT-induced effects on cyclic adenosine monophosphate (cAMP) formation in h5-ht5A-HEK 293 cells.
Section snippets
Materials
The h5-ht5A-HEK 293 cell line, stably expressing the human 5-ht5A receptor, was obtained from BioResearch Ireland (Dublin, Ireland). 5-Carboxamido[]tryptamine ([]5-CT, 50–100 Ci/mmol), [N-methyl-]lysergic acid diethylamide ([]LSD, 60–86 Ci/mmol) and guanosine-5′[γ-]thiotriphosphate ([]GTPγS, >1000 Ci/mmol) were purchased from Amersham Pharmacia Biotech (Little Chalfont, UK). [2,8-]Adenosine 3′,5′-cyclic phosphate ([]cAMP, 25–40 Ci/mmol) and [2,8-]adenine ([]adenine), 20–40
Binding of []5-CT and []LSD to h5-ht5A-HEK 293 cell membranes
The h5-ht5A receptor binding properties were investigated using []5-CT and []LSD binding to membranes of h5-ht5A-HEK 293 cells. The []5-CT concentration-binding isotherms were best fitted to a two-binding-site model using non-linear regression (GraphPad Prism program, F test, P<0.005) (Fig. 1-A). The biphasic Scatchard plot, shown in Fig. 1-A (inset), clearly demonstrated the presence of both high- and low-affinity sites. Mean Kd and Bmax values from curve fitting to a one-binding-site
Discussion
We have investigated the binding and the G protein coupling properties of the h5-ht5A receptor expressed in stably transfected HEK 293 cells and have demonstrated h5-ht5A receptor-mediated G protein activation and inhibition of adenylate cyclase. Previously reported expression studies in COS cells failed to demonstrate coupling of the h5-ht5A receptor to G proteins (Rees et al., 1994; Grailhe et al., 1995). During the preparation of this manuscript, however, functional coupling to G proteins
Acknowledgements
This work was supported by a grant from the IWT (Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie). The scientific advice of Katty Josson is much appreciated. We thank Walter Gommeren, Paul Van Gompel, Ria Wouters, Danielle van de Wiel and Martine Ercken for their helpful practical advice.
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