Peter Vanhoenacker

5-HT7 receptors: current knowledge and future prospects

Identification of three splice variants of the 5-HT7 receptor suggests a possible diversity in 5-HT7 receptor action. Indeed, 5-HT7 receptors have been implicated in the pathophysiology of several disorders; they play a role in smooth muscle relaxation within the vasculature and in the gastrointestinal tract. However, most of these assignments are derived from receptor localization studies and investigations using nonselective ligands, and are therefore mainly suggestive. The development of selective 5-HT7 receptor antagonists will be of utmost importance in determining the actual physiological and pharmacological roles of this receptor. Major challenges of 5-HT7 receptor research are determination of the transcriptional regulation of the gene encoding the 5-HT7 receptor and elucidation of the differences in regulation and signalling of its four gene products.

Role of the 5-HT7 receptor in the central nervous system: from current status to future perspectives.

Pharmacological and genetic tools targeting the 5-hydroxytryptamine (5-HT)7 receptor in preclinical animal models have implicated this receptor in diverse (patho)physiological processes of the central nervous system (CNS). Some data obtained with 5-HT7 receptor knockout mice, selective antagonists, and, to a lesser extent, agonists, however, are quite contradictory. In this review, we not only discuss in detail the role of the 5-HT7 receptor in the CNS but also propose some hypothetical models, which could explain the observed inconsistencies. These models are based on two novel concepts within the field of G protein-coupled receptors (GPCR), namely biphasic signaling and G protein-independent signaling, which both have been shown to be mediated by GPCR dimerization. This led us to suggest that the 5-HT7 receptor could reside in different dimeric contexts and initiate different signaling pathways, depending on the neuronal circuitry and/or brain region. In conclusion, we highlight GPCR dimerization and G protein-independent signaling as two promising future directions in 5-HT7 receptor research, which ultimately might lead to the development of more efficient dimer- and/or pathway-specific therapeutics.

BTB Protein KLHL12 Targets the Dopamine D4 Receptor for Ubiquitination by a Cul3-based E3 Ligase

Dopamine receptors belong to the superfamily of G-protein-coupled receptors and are subdivided into D1-type (D1 and D5) and D2-type (D2, D3, and D4) receptors. The D4 receptor has a remarkable polymorphism in its third intracellular loop, which is under intensive investigation and which has been associated with, among other conditions, attention deficit hyperactivity disorder. Here, we demonstrate that KLHL12, a BTB-Kelch protein, specifically binds to this polymorphic region of the D4 receptor through its Kelch domain. Moreover, we show that KLHL12 also interacts with Cullin3 and thereby functions as an adaptor to target the D4 receptor to an E3 ubiquitin ligase complex. By ubiquitination assays in eukaryotic cells, we further demonstrate that overexpression of KLHL12 strongly promotes ubiquitination of the D4 receptor. In addition, we show that also other dopamine receptor subtypes undergo basal ubiquitination, but this is not affected by KLHL12. These data are the first to show ubiquitination of dopamine receptors and the first to identify a protein specifically interacting with the D4 polymorphism, thereby building up an E3 ligase complex with substrate specificity toward the D4 receptor.

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.

Agonistic properties of alniditan, sumatriptan and dihydroergotamine on human 5-HT1B and 5-HT1D receptors expressed in various mammalian cell lines

Alniditan, a novel migraine abortive agent, is a potent 5‐HT1B/5‐HT1D receptor agonist of nM affinity. We compared the agonistic properties of alniditan, sumatriptan and dihydroergotamine on the cloned human 5‐HT1B receptor expressed at 200 fmol mg−1 protein (Bmax) in non‐induced L929sA cells, at 740 fmol mg−1 protein in HEK 293 and at 2300 fmol mg−1 protein in mIFNβ‐induced L929sA cells, and on the human cloned 5‐HT1D receptor expressed in C6 glioma cells (Bmax 780 fmol mg−1 protein). Sodium butyrate treatment increased the expression level of human (h)5‐HT1B receptors in HEK 293 cells and h5‐HT1D receptors in C6 glioma cells approximately 3 fold, the binding affinities of [3H]‐5‐HT and [3H]‐alniditan were unaffected. Agonistic properties were evaluated based on inhibition of cyclic AMP accumulation in the cells after stimulation of adenylyl cyclase by forskolin or isoproterenol. Alniditan, sumatriptan and dihydroergotamine were full agonists at the h5‐HT1B receptor (IC50 values were 1.7, 20 and 2 nM, respectively in HEK 293 cells) and h5‐HT1D receptors (IC50 values of 1.3, 2.6 and 2.2 nM, respectively). At the h5‐HT1B receptor the agonist potency of the compounds slightly increased with higher receptor density. The opposite was seen for antagonists (ocaperidone, risperidone and ritanserin). This comparative study demonstrated that alniditan was 10 times more potent than sumatriptan at the h5‐HT1B receptor, and twice as potent at the h5‐HT1D receptor. Dihydroergotamine was more potent an agonist at the h5‐HT1B receptor when expressed at high and low level in L929sA cells (but not in HEK 293 cells), and was less potent at the h5‐HT1D receptor.

Evaluation of the tetracycline- and ecdysone-inducible systems for expression of neurotransmitter receptors in mammalian cells.

Establishing a stable cell line that expresses a particular protein of interest is often a laborious and time‐consuming experience. With constitutive expression systems, a gradual loss of the highly expressing clones over a given time span and/or a severe counter‐selection due to toxicity of the expressed protein for the host cell line are major drawbacks. In both cases, inducible expression systems offer a valuable alternative. Over the years, many regulated expression systems have been developed and evaluated. In the present study, we compare the efficiency, the advantages and the drawbacks of a tetracycline‐ and an ecdysone‐inducible system for expression of the reporter protein chloramphenicol acetyltransferase and of different G‐protein‐coupled serotonin (5‐HT) receptors. A high level of expression of different 5‐HT receptors was obtained with the tetracycline‐inducible system. In the cell line L929, which stably expresses the tetracycline‐responsive transactivator, a maximum ligand binding of 20 000 and 9500 fmol/mg protein was measured for the h5‐HT1B and h5‐ht1F receptors, respectively. In the HEK293rtTA cell line, levels of 15 700, 3000, and 9100 fmol bound ligand/mg protein were obtained for the h5‐HT1B, h5‐ht1F and h5‐HT4b receptors, respectively. These high expression levels remained stable for several months of continuous culture. Although the ecdysone‐inducible expression system was useful for tightly regulated expression, the levels were far lower than those obtained with the tetracycline system (e.g. 640 fmol bound ligand/mg protein for the h5‐ht1F receptor in HEK293EcR).

Resistance of the dopamine D4 receptor to agonist-induced internalization and degradation

Dopamine receptors are G-protein-coupled receptors involved in the control of motivation, learning, and fine-tuning of motor movement, as well as modulation of neuroendocrine signalling. Stimulation of G-protein-coupled receptors normally results in attenuation of signalling through desensitization, followed by internalization and down-regulation of the receptor. These processes allow the cell to regain homeostasis after exposure to extracellular stimuli and offer protection against excessive signalling. Here, we have investigated the agonist-mediated attenuation properties of the dopamine D4 receptor. We found that several hallmarks of signal attenuation such as receptor phosphorylation, internalization and degradation showed a blunted response to agonist treatment. Moreover, we did not observe recruitment of beta-arrestins upon D4 receptor stimulation. We also provide evidence for the constitutive phosphorylation of two serine residues in the third intracellular loop of the D4 receptor. These data demonstrate that, when expressed in CHO, HeLa and HEK293 cells, the human D4 receptor shows resistance to agonist-mediated internalization and down-regulation. Data from neuronal cell lines, which have been reported to show low endogenous D4 receptor expression, such as the hippocampal cell line HT22 and primary rat hippocampal cells, further support these observations.

KLHL12-mediated ubiquitination of the dopamine D4 receptor does not target the receptor for degradation

In previous studies, we identified KLHL12 as a novel interaction partner of the dopamine D4 receptor that functions as an adaptor in a Cullin3-based E3 ubiquitin ligase complex to target the receptor for ubiquitination. In this study, we show that KLHL12 promotes poly-ubiquitination of the receptor by performing ubiquitination assays in eukaryotic cells. Furthermore, we demonstrate that KLHL12 not only interacts with both immature, ER-associated and mature, plasma membrane-associated D4 receptors, but also promotes ubiquitination of both receptor subpools. Unexpectedly, however, KLHL12-mediated receptor ubiquitination does not promote proteasomal degradation of newly synthesized receptors through the ER-associated degradation pathway or lysosomal degradation of mature receptors. Moreover, our data reveal that D4 receptors do not undergo agonist-promoted ubiquitination or degradation, in contrast to many other G-protein-coupled receptors (GPCRs) indicating that ubiquitination of GPCRs does not defaultly lead to receptor degradation. Interestingly, KLHL12 does also interact with beta-arrestin2 but this has no effect on the ubiquitination or localization of beta-arrestin2 nor on the internalization of the D4 receptor.

Molecular integrity and usefulness of episomal expression vectors derived from BK and Epstein-Barr virus.

High-level and stable production of a protein of interest is one of the most important parameters when considering the development of an efficient vector system for heterologous gene expression. In order to achieve this goal, we have used episomal vector elements derived from Epstein-Barr virus (EBV) or BK virus (BKV) in combination with the strictly regulated interferon-inducible Mx promoter. Here we demonstrate that EBV-derived vectors replicate efficiently in all cell lines tested (i.e. HEK293, HeLaH21 and Vero), yielding stable transfectants with a high, inducible expression level and almost no background. In contrast, BKV-derived vectors are much more restricted to particular cell types and hampered by DNA rearrangements, which is a serious drawback for use over a longer timespan.

Inducible production of recombinant human Flt3 ectodomain variants in mammalian cells and preliminary crystallographic analysis of Flt3 ligand-receptor complexes

The extracellular complex between the haematopoietic receptor Flt3 and its cytokine ligand (FL) is the cornerstone of signalling cascades that are central to early haematopoiesis and the immune system. Here, efficient protocols for the production of two ectodomain variants of human Flt3 receptor, Flt3D1-D5 and Flt3D1-D4, for structural studies are reported based on tetracycline-inducible stable cell lines in HEK293S cells deficient in N-acetylglycosaminyltransferase I (GnTI-/-) that can secrete the target proteins with limited and homogeneous N-linked glycosylation to milligram amounts. The ensuing preparative purification of Flt3 receptor-ligand complexes yielded monodisperse complex preparations that were amenable to crystallization. Crystals of the Flt3D1-D4-FL and Flt3D1-D5-FL complexes diffracted to 4.3 and 7.8 Å resolution, respectively, and exhibited variable diffraction quality even within the same crystal. The resulting data led to the successful structure determination of Flt3D1-D4-FL via a combination of molecular-replacement and density-modification protocols exploiting the noncrystallographic symmetry and high solvent content of the crystals.