5-HT2A receptor signalling through phospholipase D1 associated with its C-terminal tail

The 5-HT2AR (5-hydroxytryptamine-2A receptor) is a GPCR (G-protein-coupled receptor) that is implicated in the actions of hallucinogens and represents a major target of atypical antipsychotic agents. In addition to its classical signalling though PLC (phospholipase C), the receptor can activate several other pathways, including ARF (ADP-ribosylation factor)-dependent activation of PLD (phospholipase D), which appears to be achieved through a mechanism independent of heterotrimeric G-proteins. In the present study we show that wild-type and inactive constructs of PLD1 (but not PLD2) respectively facilitate and inhibit ARF-dependent PLD signalling by the 5-HT2AR. Furthermore we demonstrate that PLD1 specifically co-immunoprecipitates with the receptor and binds to a distal site in GST (glutathione transferase) fusion protein constructs of its C-terminal tail which is distinct from the ARF-interaction site, thereby suggesting the existence of a functional ARF-PLD signalling complex directly associated with this receptor. This reveals the spatial co-ordination of an important GPCR, transducer and effector into a physical complex that is likely to reinforce the impact of receptor activation on a heterotrimeric G-protein-independent signalling pathway. Signalling of this receptor through such non-canonical pathways may be important to its role in particular disorders

LSD but not lisuride disrupts prepulse inhibition in rats by activating the 5-HT2A receptor

Compounds that activate the 5-HT2A receptor, such as lysergic acid diethylamide (LSD), act as hallucinogens in humans. One notable exception is the LSD congener lisuride, which does not have hallucinogenic effects in humans even though it is a potent 5-HT2A agonist. LSD and other hallucinogens have been shown to disrupt prepulse inhibition (PPI), an operational measure of sensorimotor gating, by activating 5-HT2A receptors in rats. We tested whether lisuride disrupts PPI in male Sprague–Dawley rats. Experiments were also conducted to identify the mechanism(s) responsible for the effect of lisuride on PPI and to compare the effects of lisuride to those of LSD. Confirming a previous report, LSD (0.05, 0.1, and 0.2 mg/kg, s.c.) reduced PPI, and the effect of LSD was blocked by pretreatment with the selective 5-HT2A antagonist MDL 11,939. Administration of lisuride (0.0375, 0.075, and 0.15 mg/kg, s.c.) also reduced PPI. However, the PPI disruption induced by lisuride (0.075 mg/kg) was not blocked by pretreatment with MDL 11,939 or the selective 5-HT1A antagonist WAY-100635 but was prevented by pretreatment with the selective dopamine D2/D3 receptor antagonist raclopride (0.1 mg/kg, s.c). The effect of LSD on PPI is mediated by the 5-HT2A receptor, whereas activation of the 5-HT2A receptor does not appear to contribute to the effect of lisuride on PPI. These findings demonstrate that lisuride and LSD disrupt PPI via distinct receptor mechanisms and provide additional support for the classification of lisuride as a non-hallucinogenic 5-HT2A agonist

Elucidation of the serotonin 5-HT(2A) receptor-coupled phospholipase A(2) signaling pathway

The purpose of the present study was to employ pharmacological and biochemical tools to explore the ability of the serotonin2A (5-HTZA) receptor to couple to multiple signaling cascades, namely the phospholipase C (PLC) and phospholipase A2 (PLA2) pathways, and particularly to elucidate the mechanism by which the 5-HTZA receptor activates PLA 2. Initial studies were conducted to verify that PLA2 activation was receptor-mediated and not subsequent to PLC activation. The data suggest that the 5HT2A receptor is able to activate both signaling pathways independently because several specific inhibitors of signaling molecules along each cascade were employed with no evidence that they could modulate signaling in the other pathway. Next, the ability of structurally distinct ligands to activate both the PLC and PLA2 signaling pathways was examined. In support of the hypothesis of agonist-directed trafficking, many 5-HT 2A receptor agonists displayed differential activation of the PLC and PLA2 signaling pathways. Furthermore, many of these agonists showed a difference in potency between PLC and PLA2 pathway activation, which is consistent with receptor-inactivation studies that suggested the existence of pathway-specific receptor reserve. Having verified that 5-HT2A receptor-mediated PLA activation was independent of PLC activation, studies were then conducted to elucidate a potential signal transduction cascade responsible for linking 5-HT 2A receptor activation to AA liberation. By employing various selective inhibitors, toxins, and antagonistic peptide constructs, the 5HT2A receptor was demonstrated to couple to PLA2 activation through a complex signaling mechanism involving both Gαi/o-associated Gβγ-mediated ERK1,2 activation and Gα12/13-coupled Rho-mediated p38 activation. Thus, these results suggest that the 5-HT 2A receptor can couple to multiple G proteins, namely PLC-coupled Gα q, PLA2-coupled Gαi/o, and PLA2-coupled Gα12/13, to stimulate PLA2-AA release and PLC-IP accumulation through largely independent signaling pathways. Overall, these studies demonstrate not only the ability, but also a potential mechanism by which the 5-HT2A receptor can couple to multiple signaling cascades. The data presented herein support the findings of other laboratories, which together suggest GPCR signaling to be more complex that thought previously. An understanding of the signal transduction pathways that are activated by a specific drug will provide insight into the physiological response mediated by its receptor

Arresting serotonin

[No abstract available

Agonist-directed signaling of the serotonin 2A receptor depends on β-arrestin-2 interactions in vivo

Visual and auditory hallucinations accompany certain neuropsychiatric disorders, such as schizophrenia, and they also can be induced by the use or abuse of certain drugs. The heptahelical serotonin 2A receptors (5-HT2ARs) are molecular targets for drug-induced hallucinations. However, the cellular mechanisms by which the 5-HT2AR mediates these effects are not well understood. Drugs acting at the 5-HT2AR can trigger diverse signaling pathways that may be directed by the chemical properties of the drug. β-arrestins are intracellular proteins that bind to heptahelical receptors and represent a point where such divergences in ligand-directed functional signaling could occur. Here we compare the endogenous agonist, serotonin, to a synthetic 5-HT2AR hallucinogenic agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI), in mice lacking β-arrestin-2, as well as in cells lacking β-arrestins. In mice, we find that serotonin induces a head twitch response by a β-arrestin-2-dependent mechanism. However, DOI invokes the behavior independent of β-arrestin-2. The two structurally distinct agonists elicit different signal transduction and trafficking patterns upon activation of 5-HT2AR, which hinge on the presence of β-arrestins. Our study suggests that the 5-HT2AR–β-arrestin interaction may be particularly important in receptor function in response to endogenous serotonin levels, which could have major implications in drug development for treating neuropsychiatric disorders such as depression and schizophrenia