Project summary: Serotonin, or 5 hydroxytryptamine (5HT) is an endogenous transmitter that is broadly implicated in many modalities of human behavior including mood, libido, appetite and sleep, as well as peripheral functions in platelet aggregation, immune response and bone density. In the brain, this single transmitter binds and activates seven different families of 5HT receptors with five distinct modes of signal transduction, including all four classes of G protein-coupled receptor (GPCR) (Gi, Gs, Gq and G12/13) and a ligand-gated ion channel. Alterations in the relative balance of these diverse signaling pathways by 5HT has been implicated in a plethora of syndromes including depression, major depressive disorder, bipolar disorder, schizophrenia, obsessive compulsive disorder and attention deficit hyperactivity disorder. Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed for the treatment of depression, and more than 15% of adults in the US have taken an antidepressant within the past 12 months. In theory, the efficacy of these drugs is due to their ability to increase 5HT levels by blocking reuptake of 5HT. However, while these drugs increase 5HT levels in as little as 2 hours, it takes 4-8 weeks for them to become effective at ameliorating depression. This suggests that the mechanism of action of SSRIs is more complicated than simple increases in transmitter levels. We hypothesize that high levels of 5HT, produced through use of SSRIs, can, in time, rebalance the expression levels of the various 5HT receptors and, by doing so, provide therapeutic benefit. Specifically, we propose that some of the 5HTRs, when activated by 5HT, undergo endocytosis and recycling/resensitization while others are endocytosed and degraded in the lysosome. The high levels of 5HT provided by the SSRI can, thereby, drive down expression of certain subtypes of 5HTR, while maintaining a high level of signaling through others--in effect “rebalancing” 5HT signal transduction to more closely resemble the non-depressed state. Here, we will perform a comprehensive analysis of the endocytic and post-endocytic properties of the entire family of 5HTRs. Remarkably, there is very limited knowledge regarding the post-endocytic sorting properties of the 5HTRs. Hence, regardless of outcome, these data will provide new information regarding how increased 5HT levels, due to SSRIs, could alter receptor expression and could inform new approaches to serotonin therapeutics designed to rebalance 5HT signal transduction.