Regulation of Serotonin 1B Receptor by Glycogen Synthase Kinase-3

Abstract

In response to 5-hydroxytryptamine (5-HT), the type 1 serotonin receptors (5-HT1Rs) preferentially couple to the inhibitory G protein and elicit many physiological and behavioral processes. However, their regulation by intracellular protein kinases has not been fully investigated. In this study, we identified that glycogen synthase kinase-3 (GSK3) differentially regulates 5-HT1Rs. In receptor-expressing cells and brain slices, activation of both 5-HT1AR and 5-HT1BR reduced forskolin-stimulated cAMP production, but only the effect of 5-HT1BR was abolished by selective GSK3 inhibitors, deletion of GSK3␤ by RNAi, or overexpression of impaired GSK3␤ mutants (R96A and K85,86A). A consensus GSK3 phosphorylation sequence was identified between the serine-154 and threonine-158 in the second intracellular loop of 5-HT1BR. Mutation of either serine-154 or threonine-158 to alanine significantly reduced response of 5-HT1BR to 5-HT. Active GSK3␤ interacted with resting 5-HT1BR to form a protein complex. The interaction was enhanced by receptor activation, abolished by GSK3 inhibitors, and dependent on the phosphorylation state of serine-154. In addition, regulation of 5-HT1BR by GSK3 changed the dynamics of agonist-induced cell surface receptor internalization, in which lack of phosphorylation at Ser154 resulted in sustained reduction of 5-HT1BR at the cell surface. Although the physiological consequences of selective regulation of 5-HT1BR by GSK3 remain to be identified, findings in this study reveal a new function of GSK3 as a protein kinase that is able to selectively regulate G protein-coupled receptors.Most serotonin (5-HT) receptors are G protein-coupled receptors (GPCRs) that are classified by their sequence homology and by the type of G proteins and signal transduction pathways with which they are associated (Hannon and Hoyer, 2008). The type 1 5-HT receptors (5-HT1Rs), including subtypes A (5-HT1AR) and B (5-HT1BR), are mainly coupled to the inhibitory G protein, which links the receptors to inhibition of adenylyl cyclase and regulation of several other signal pathways (Raymond et al., 2001). 5-HT1AR and 5-HT1BR are found in several brain areas, such as the dorsal raphe nucleus, nucleus accumbens, cerebral cortex, hippocampus, and striatum. In the raphe nucleus, 5-HT1AR and 5-HT1BR are characterized as autoreceptors, with the former located at the somatodendritic area and the latter at the axon terminus. In many other brain regions and peripheral tissues, 5-HT1AR and 5-HT1BR also function as heteroreceptors in nonserotonergic neurons (Hannon and Hoyer, 2008). 5-HT1Rs are involved in many physiological processes in the brain, especially regulating turnover of 5-HT and other neurotransmitters in several brain regions (Ase et al., 2000). 5-HT1Rs are a major group of serotonin receptors that modulate behaviors, especially those relevant to neuropsychiatric diseases, such as anxiety, mood, and aggression (Gingrich and Hen, 2001). Therefore, proper regulation of activities of 5-HT1Rs is crucially important in main...

A role for Akt and glycogen synthase kinase-3 as integrators of dopamine and serotonin neurotransmission in mental health

“…80 Furthermore, there is strong evidence for a role of GSK3 in the regulation of circadian rhythm, epigenetic gene regulation 29 and 5-HT 1B receptor cell surface trafficking. 113,114 Another intriguing observation is that changes in the regulation of Akt and GSK3 activity so far are similar across different drug categories. Overall, antipsychotics, antidepressants acting on 5-HT neurotransmission and lithium have all been reported to activate Akt and inhibit GSK3 in vivo.…”

Fine-Tuning of the RIG-I-Like Receptor/Interferon Regulatory Factor 3-Dependent Antiviral Innate Immune Response by the Glycogen Synthase Kinase 3/β-Catenin Pathway

“…As pharmacological inhibitors are prone to off-target effects, we used the GSK-3 inhibitors at low concentrations (2 to 5 M). Other studies have used CHIR (57)(58)(59)(60)(61), BIO (62,63), and BIO-ac (38,64) at concentrations as high as 10 to 20 M. Moreover, reconstitution of DKO ES cells with kinase-dead forms of GSK-3␣ and GSK-3␤ showed decreased antiviral mRNA expression compared to that obtained by reconstitution with WT forms. Although we do not completely rule out a kinase-independent function for GSK-3 in innate immunity, our data obtained with DKO ES cells and GSK-3 inhibitors (Fig.…”

Glycogen Synthase Kinase-3β Is a Functional Modulator of Serotonin-1B Receptors

“…Human G i ␣ 2 and human ␤-arrestin2 cDNAs were subcloned into pEYFP-C1 with N-terminal YFP for G i ␣ 2 and C-terminal YFP for ␤-arrestin2. Mutagenesis of mouse 5-HT1BR was performed as described previously (Chen et al, 2009). All constructs were verified by DNA sequencing before cellular transfection.…”

“…The level of cAMP was measured in CHO cells using an enzyme immunoassay kit (Direct BioTrak; Amersham/GE Healthcare) (Chen et al, 2009). To minimize the potential nonselective effects on GSK3 by other inhibitors, all experiments with cAMP measurement in this study were conducted in the absence of phosphodiesterase inhibitors.…”

“…We found previously that GSK3␤ selectively interacts with serotonin 1B receptors (5-HT1BR) (Chen et al, 2009). 5-HT1BR belong to the Class A G protein-coupled receptors (GPCRs) that activate the inhibitory G protein (G i ) to inhibit adenylyl cyclase and cAMP production (Raymond et al, 2001) and to activate trophic factor-regulated signaling, such as Akt (Leone et al, 2000).…”