Characterization of serotonin-5-HTR1E signaling pathways and its role in cell survival

Abstract

5-Hydroxy tryptamine receptor 1E (5-HTR1E) is reported to activate cAMP and ERK pathways via its ligands and binding partners, but the detailed mechanism underlying the serotonin induced 5-HTR1E signaling is still not known. In the present study, we determined the cellular regulators of ERK and cAMP signaling pathways in response to serotonin induced 5-HTR1E activation in 5-HTR1E overexpressing HEK293 cells. We found that Pertussis Toxin (PTX) treatment completely reversed the effect of serotonin-5-HTR1E mediated signaling on cAMP and ERK pathways, confirming the involvement of a Gαi-linked cascade. We also observed that Gβγ and Gq were not associated with 5-HTR1E activation, while blocking PKA inhibited ERK signaling only, and had no effect on cAMP. Additionally, serotonin-stimulated ERK1/2 phosphorylation was similar in 5-HTR1E overexpressing, β-arrestin-deficient HEK293 cells and is solely dependent on G protein signaling. siRNA mediated gene knockout studies in SH-SY5Y cells revealed that the inhibition of 5-HTR1E reduced the expression of cMyc, Cyclin D1, Cyclin E and BCL2 genes which are related to cell cycle regulation and survival. MTT assays showed that 5-HTR1E knockdown in SHSY-5Y and U118 cells inhibited cell survival significantly. In addition to the signaling mechanism, we also performed RNA-seq analysis in 5-HTR1E overexpressing HEK293 cells and found that 5-HTR1E can regulate the expression of Receptor activity modifying protein 1 (RAMP1), Nuclear receptor 1 (NR4A1) and other Cyclin genes. These findings indicate that serotonin interaction with 5-HTR1E receptor simultaneously activates cAMP and ERK pathway in HEK293 cells and its expression is important for cell survival.

Structural insights into the unexpected agonism of tetracyclic antidepressants at serotonin receptors 5-HT 1e R and 5-HT 1F R

“…of recruiting arrestin to terminate G protein signaling or engage in noncanonical signaling (14). Thus, to test potential agonist activity of aminergic drugs at 5-HT 1e R and 5-HT 1F R, we measured receptormediated reduction in cellular cAMP using the GloSensor biosensor.…”

“…While this oversight contributes to our poor understanding of 5-HT 1e R and 5-HT 1F R's pharmacological and molecular mechanisms, examining the activity of serotonergic drugs may uncover physiological (side) effects of drugs mediated by these receptors. Moreover, such activity could potentially be harnessed to explore pharmacophores that yield receptor-selective probes, such as first in class 5-HT 1e R-selective tool compounds with which to explore previous suggestions of 5-HT 1e R as a viable pharmacological target in the treatment of certain types of cancers (13,14).…”

Structural Insights into the Unexpected Agonism of Tetracyclic Antidepressants at Serotonin Receptors 5-HT1eR and 5-HT1FR

“…While this oversight contributes to our poor understanding of 5-HT 1e R’s and 5-HT 1F R’s pharmacological and molecular mechanisms, examining the activity of serotonergic drugs may uncover physiological (side) effects of drugs mediated by these receptors. Moreover, such activity could potentially be harnessed to explore pharmacophores that yield receptor-selective probes, such as first in class 5-HT 1e R-selective tool compounds with which to explore previous suggestions of 5-HT 1e R as a viable pharmacological target in the treatment of certain types of cancers ( 11, 12 ).…”