Kjetil Wessel Andressen

The serotonin 5-HT7 receptors: two decades of research

Abstract Like most neurotransmitters, serotonin possesses a simple structure. However, the pharmacological consequences are more complex and diverse. Serotonin is involved in numerous functions in the human body including the control of appetite, sleep, memory and learning, temperature regulation, mood, behavior, cardiovascular function, muscle contraction, endocrine regulation, and depression. Low levels of serotonin may be associated with several disorders, namely increase in aggressive and angry behaviors, clinical depression, Parkinson’s disease, obsessive–compulsive disorder, eating disorders, migraine, irritable bowel syndrome, tinnitus, and bipolar disease. These effects are mediated via different serotonin (5-HT) receptors. In this review, we will focus on the last discovered member of this serotonin receptor family, the 5-HT7 receptor. This receptor belongs to the G protein-coupled receptor superfamily and was cloned two decades ago. Later, different splice variants were described but no major functional differences have been described so far. All 5-HT7 receptor variants are coupled to Gαs proteins and stimulate cAMP formation. Recently, several interacting proteins have been reported, which can influence receptor signaling and trafficking.

Substrate Specificities of G Protein-Coupled Receptor Kinase-2 and -3 at Cardiac Myocyte Receptors Provide Basis for Distinct Roles in Regulation of Myocardial Function

The closely related G protein-coupled receptor kinases GRK2 and GRK3 are both expressed in cardiac myocytes. Although GRK2 has been extensively investigated in terms of regulation of cardiac β-adrenergic receptors, the substrate specificities of the two GRK isoforms at G protein-coupled receptors (GPCR) are poorly understood. In this study, the substrate specificities of GRK2 and GRK3 at GPCRs that control cardiac myocyte function were determined in fully differentiated adult cardiac myocytes. Concentration-effect relationships of GRK2, GRK3, and their respective competitive inhibitors, GRK2ct and GRK3ct, at endogenous endothelin, α1-adrenergic, and β1-adrenergic receptor-generated responses in cardiac myocytes were achieved by adenovirus gene transduction. GRK3 and GRK3ct were highly potent and efficient at the endothelin receptors (IC50 for GRK3, 5 ± 0.7 pmol/mg of protein; EC50 for GRK3ct, 2 ± 0.2 pmol/mg of protein). The α1-adrenergic receptor was also a preferred substrate of GRK3 (IC50,7 ± 0.4 pmol/mg of protein). GRK2 lacked efficacy at both endothelin and α1-adrenergic receptors despite massive overexpression. On the contrary, both GRK2ct and GRK3ct enhanced β1-adrenergic receptor-induced cAMP production with comparable potencies. However, the potency of GRK3ct at β1-adrenergic receptors was at least 20-fold lower than that at endothelin receptors. In conclusion, this study demonstrates distinct substrate specificities of GRK2 and GRK3 at different GPCRs in fully differentiated adult cardiac myocytes. As inferred from the above findings, GRK2 may play its primary role in regulation of cardiac contractility and chronotropy by controlling β1-adrenergic receptors, whereas GRK3 may play important roles in regulation of cardiac growth and hypertrophy by selectively controlling endothelin and α1-adrenergic receptors.

Disheveled regulates precoupling of heterotrimeric G proteins to Frizzled 6

Frizzleds (FZDs) are classified as G‐protein‐coupling receptors, but how signals are initiated and specified through heterotrimeric G proteins is unknown. FZD6 regulates convergent extension movements, and its C‐terminal Arg511Cys mutation causes nail dysplasia in humans. We investigated the functional relationship between FZD6, Disheveled (DVL), and heterotrimeric G proteins. Live cell imaging combined with fluorescence recovery after photobleaching (FRAP) revealed that inactive human FZD6 precouples to Gαi1 and Gαq but not to GαoA,Gαs, and Gα12 proteins. G‐protein coupling is measured as a 10–20% reduction in the mobile fraction of fluorescently tagged G proteins on chemical receptor surface cross‐linking. The FZD6 Arg511Cys mutation is incapable of G‐protein precoupling, even though it still binds DVL. Using both FRAP and Förster resonance energy transfer (FRET) technology, we showed that the FZD6‐Gαi1 and FZD‐Gαq complexes dissociate on WNT‐5A stimulation. Most important, G‐protein precoupling of FZD6 and WNT‐5A‐induced signaling to extracellular signal‐regulated kinase1/2 were impaired by DVL knockdown or overexpression, arguing for a strict dependence of FZD6‐G‐protein coupling on DVL levels and identifying DVL as a master regulator of FZD/G‐protein signaling. In summary, we propose a mechanistic connection between DVL and G proteins integrating WNT, FZD, G‐protein, and DVL function.—Kilander, M. B. C., Petersen, J., Andressen, K. W., Ganji, R. S. Levy, F. O., Schuster, J., Dahl N., Bryja, V., Schulte, G. Disheveled regulates precoupling of heterotrimeric G proteins to Frizzled 6. FASEB J. 28, 2293–2305 (2014). www.fasebj.org

Unaltered Agonist Potency upon Inducible 5-HT 7(a) but not 5-HT 4(b) Receptor Expression Indicates Agonist-Independent Association of 5-HT 7(a) Receptor and G s

We compared adenylyl cyclase (AC) activation by the G protein-coupled human serotonin (5-HT) receptors 5-HT4(b) and 5-HT7(a) using an ecdysone-inducible expression system, which allowed for reproducible expression of increasing receptor densities in clonal HEK293 (EcR293) cell lines. Low constitutive expression of receptors (2-70 fmol/mg protein) was observed and could be titrated up to 50-200-fold (approximately 400-7000 fmol/mg protein) by the ecdysone analogue ponasterone A. Although 5-HT-stimulated AC activity increased with receptor density, interclonal variation precluded comparisons of coupling efficiency. Interestingly, the potency of 5-HT to stimulate AC increased with increasing receptor density only in clones expressing 5-HT4(b) receptors. The potency for 5-HT did not change in clones expressing 5-HT7(a) receptors, even though 5-HT-stimulated AC activity approached asymptotic levels. This indicates that potency of 5-HT for stimulation of AC through the 5-HT7(a) receptor is independent of receptor-Gs stoichiometry and is consistent with a model where the 5-HT7(a) receptors are tightly associated with G protein, independent of agonist binding. This supports the existence of a complex between inactive receptor and G protein, as predicted by the cubic ternary complex model. In such a system, spare receptors do not lead to increased potency of an agonist with increased receptor density.

Activation of Adenylyl Cyclase by Endogenous Gs-Coupled Receptors in Human Embryonic Kidney 293 Cells Is Attenuated by 5-HT7 Receptor Expression

Human 5-hydroxytryptamine7 (5-HT7) receptors display characteristics shared with receptors believed to form a tight physical coupling with G protein in the absence of ligand. Some receptors apparently preassociated with Gi/o and Gq/11 are reported to inhibit the signaling of other similarly coupled G protein-coupled receptors by limiting their access to activate a common G protein pool. Therefore, we determined whether 5-HT7 receptor expression was sufficient to limit signaling of endogenously expressed Gs-coupled receptors in human embryonic kidney (HEK) 293 cells. Using the ecdysone-inducible expression system, which allows for the titration of increasing receptor density in the same clonal cell line, we compared the effects of 5-HT4(b) and 5-HT7(a,b,d) receptor expression on adenylyl cyclase (AC) stimulation by the endogenous Gs-coupled β-adrenergic (βAR) and prostanoid EP (EPR) receptors. βAR- and EPR-stimulated AC activity was attenuated by 5-HT7 receptor expression in both membrane preparations and intact HEK293 cells. βAR- and EPR-stimulated AC activity was unaffected by expression of the Gs-coupled 5-HT4 receptor. The mechanism of this heterologous desensitization seems independent of protein kinase A activation, nor does it occur at the level of G protein activation because 1) βAR- and EPR-stimulated AC activity was not restored to control values when Gαs was overexpressed; and 2) β1AR and β2AR activation of Gαs was unaffected by the expression of 5-HT7 receptors. In addition, overexpression of AC isoforms was unable to rescue βAR- and EPR-stimulated AC activity. Therefore, 5-HT7 receptors probably limit access and/or impede activation of AC by βAR and EP receptors. Although the 5-HT7 receptor may preassociate with G protein and/or AC, the mechanism of this heterologous desensitization remains elusive.

5-HT4-elicited positive inotropic response is mediated by cAMP and regulated by PDE3 in failing rat and human cardiac ventricles

The left ventricle in failing hearts becomes sensitive to 5‐HT parallelled by appearance of functional Gs‐coupled 5‐HT4 receptors. Here, we have explored the regulatory functions of phosphodiesterases in the 5‐HT4 receptor‐mediated functional effects in ventricular muscle from failing rat and human heart.

The heterotrimeric G-protein α-subunit Gαq regulates TCR-mediated immune responses through an Lck-dependent pathway

Here, we examined the functional involvement of heterotrimeric G‐proteins in TCR‐induced immune responses. TCR/CD3 crosslinking resulted in activation of both Gαq and Gαs, but not Gαi‐2. Targeting of Gαs, Gαi‐2 and Gαq using siRNA demonstrated a specific role of Gαq in TCR signaling. Jurkat TAg T cells with Gαq knockdown displayed reduced activation of Lck and LAT phosphorylation, but paradoxically showed sustained ERK1/2 phosphorylation and increased NFAT‐AP‐1‐reporter activity implicating Gαq in the negative control of downstream signaling and IL‐2‐promoter activity. Primary T cells isolated from Gαq‐deficient mice had a similar TCR signaling response with reduced proximal LAT phosphorylation, sustained ERK1/2 phosphorylation and augmented immune responses including increased secretion of IL‐2, IL‐5, IL‐12 and TNF‐α. The effects on NFAT‐AP‐1‐reporter activity were sensitive to the Src family kinase inhibitor PP2 and were reversed by transient expression of constitutively active Lck. Furthermore, expression of constitutively active Gαq Q209L elevated Lck activity and Zap‐70 phosphorylation. Together these data argue for a role of Gαq in the fine‐tuning of proximal TCR signals at the level of Lck and a negative regulatory role of Gαq in transcriptional activation of cytokine responses.

Synthesis and pharmacological properties of novel hydrophilic 5-HT4 receptor antagonists

Serotonin (5-hydroxytryptamine, 5-HT) is an important signalling molecule in the human body. The 5-HT(4) serotonin receptor, coupled to the G protein G(s), plays important physiological and pathophysiological roles in the heart, urinary bladder, gastrointestinal tract and the adrenal gland. Both 5-HT(4) antagonists and agonists have been developed in the aim to treat diseases in these organs. 5-HT(4) agonists might have beneficial effects in the central nervous system (CNS) and therefore, 5-HT(4) antagonists might cause CNS side effects. In this study, we have developed new amphoteric 5-HT(4) antagonists. A series of cyclic indole amide derivatives possessing an oxazine ring and a piperidine alkane carboxylic acid side chain and the corresponding prodrug esters were synthesized and their binding to 5-HT(4) receptors and antagonist properties were evaluated. In addition, an indole ester without the oxazine ring and the corresponding indole amide derivatives were also tested. Octanol-water distribution (LogD(Oct7.4)) was tested for some of the synthesized ligands. The main structure-affinity characteristics of the 5-HT(4) compounds tested were that the prodrug esters show higher affinity than their corresponding free acids, indole esters show higher affinity than the corresponding amides and ligands containing the oxazine ring in the indole skeleton show higher affinity than indole derivatives not containing the ring. One representative prodrug ester and its corresponding free acid were tested for binding on a panel of receptors and showed preserved selectivity for the 5-HT(4) receptor. These new molecules may be useful to target peripheral 5-HT(4) receptors.

Cloning, genomic organization and functionality of 5-HT7 receptor splice variants from mouse brain

The serotonin (5-HT) 5-HT(7) receptors are expressed in both the central nervous system and in peripheral tissues. Receptor distribution studies and pharmacological studies have established that 5-HT(7) receptors play an important role in the control of circadian rhythms and thermoregulation. Selective 5-HT(7) receptor ligands have potential therapeutic applications for the treatment of pain and migraine, schizophrenia, anxiety, cognitive disturbances and inflammation. We have cloned two novel C-terminal splice variants of the 5-HT(7) receptor from mouse brain. These two new splice variants have almost identical sequences as the rat 5-HT(7(b)) and 5-HT(7(c)) splice variants and so were given the same name. Ligand binding assays ([(3)H]5-CT), membrane localization and functional studies in transiently transfected cells indicated that all three splice variants are well expressed on the membrane and no major differences in their respective pharmacology and their ability to activate adenylyl cyclase were observed. This is in analogy with previous reports comparing either the rat or the human variants.

Activation of adenylyl cyclase by endogenous Gs-coupled receptors in HEK293 cells is attenuated by 5-HT7 receptor expression

Human 5-hydroxytryptamine7 (5-HT7) receptors display characteristics shared with receptors believed to form a tight physical coupling with G protein in the absence of ligand. Some receptors apparently preassociated with Gi/o and Gq/11 are reported to inhibit the signaling of other similarly coupled G protein-coupled receptors by limiting their access to activate a common G protein pool. Therefore, we determined whether 5-HT7 receptor expression was sufficient to limit signaling of endogenously expressed Gs-coupled receptors in human embryonic kidney (HEK) 293 cells. Using the ecdysone-inducible expression system, which allows for the titration of increasing receptor density in the same clonal cell line, we compared the effects of 5-HT4(b) and 5-HT7(a,b,d) receptor expression on adenylyl cyclase (AC) stimulation by the endogenous Gs-coupled β-adrenergic (βAR) and prostanoid EP (EPR) receptors. βAR- and EPR-stimulated AC activity was attenuated by 5-HT7 receptor expression in both membrane preparations and intact HEK293 cells. βAR- and EPR-stimulated AC activity was unaffected by expression of the Gs-coupled 5-HT4 receptor. The mechanism of this heterologous desensitization seems independent of protein kinase A activation, nor does it occur at the level of G protein activation because 1) βAR- and EPR-stimulated AC activity was not restored to control values when Gαs was overexpressed; and 2) β1AR and β2AR activation of Gαs was unaffected by the expression of 5-HT7 receptors. In addition, overexpression of AC isoforms was unable to rescue βAR- and EPR-stimulated AC activity. Therefore, 5-HT7 receptors probably limit access and/or impede activation of AC by βAR and EP receptors. Although the 5-HT7 receptor may preassociate with G protein and/or AC, the mechanism of this heterologous desensitization remains elusive.