Background and PurposeClassically, ligands of GPCRs have been classified primarily upon their affinity and efficacy to activate a signal transduction pathway. Recent reports indicate that the efficacy of a particular ligand can vary depending on the receptor‐mediated response measured (e.g. activating G proteins, other downstream responses, internalization). Previously, we reported that inverse agonists induce both homo‐ and heterologous desensitization, similar to agonist stimulation, at the Gs‐coupled 5‐HT7 receptor. The primary objective of this study was to determine whether different inverse agonists at the 5‐HT7 receptor also induce internalization and/or degradation of 5‐HT7 receptors.Experimental ApproachHEK293 cells expressing 5‐HT7(a, b or d) receptors were pre‐incubated with 5‐HT, clozapine, olanzapine, mesulergine or SB269970 and their effects upon receptor density, AC activity, internalization, recruitment of β‐arrestins and lysosomal trafficking were measured.Key ResultsThe agonist 5‐HT and three out of four inverse agonists tested increased internalization independently of β‐arrestin recruitment. Among these, only the atypical antipsychotics clozapine and olanzapine promoted lysosomal sorting and reduced 5‐HT7 receptor density (∼60% reduction within 24 h). Inhibition of lysosomal degradation with chloroquine blocked the clozapine‐ and olanzapine‐induced down‐regulation of 5‐HT7 receptors. Incubation with SB269970 decreased both 5‐HT7(b) constitutive internalization and receptor density but increased 5‐HT7(d) receptor density, indicating differential ligand regulation among the 5‐HT7 splice variants.Conclusions and ImplicationsTaken together, we found that various ligands differentially activate regulatory processes governing receptor internalization and degradation in addition to signal transduction. Thus, these data extend our understanding of functional selectivity at the 5‐HT7 receptor.

‘Psychoneuroendocrinology’ is now quickly emerging as a hot interdisciplinary research field that addresses the interplay between neuronal and endocrine signaling in psychiatric diseases. Both glucocorticoid hormones and dopamine have an important role in maintaining normal brain functions. In this review, molecular and mechanistic aspects of glucocorticoid effects on brain function and behavior will be discussed with specific reference to dopamine signaling.

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-HT(1B) and h5-ht(1F) receptors, respectively. In the HEK293rtTA cell line, levels of 15,700, 3000, and 9100 fmol bound ligand/mg protein were obtained for the h5-HT(1B), h5-ht(1F) and h5-HT(4b) 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-ht(1F) receptor in HEK293EcR).

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.

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.

The Armadillo protein p120(ctn) associates with the cytoplasmic domain of cadherins and accumulates at cell-cell junctions. Particular Armadillo proteins such as beta-catenin and plakophilins show a partly nuclear location, suggesting gene-regulatory activities. For different human E-cadherin-negative carcinoma cancer cell lines we found expression of endogenous p120(ctn) in the nucleus. Expression of E-cadherin directed p120(ctn) out of the nucleus. Previously, we reported that the human p120(ctn) gene might encode up to 32 protein isoforms as products of alternative splicing. Overexpression of p120(ctn) isoforms B in various cell lines resulted in cytoplasmic immunopositivity but never in nuclear staining. In contrast, upon expression of p120(ctn) cDNAs lacking exon B, the isoforms were detectable within both nuclei and cytoplasm. A putative nuclear export signal (NES) with a characteristic leucine-rich motif is encoded by exon B. This sequence element was shown to be required for nuclear export and to function autonomously when fused to a carrier protein and microinjected into cell nuclei. Moreover, the NES function of endogenously or exogenously expressed p120(ctn) isoforms B was sensitive to the nuclear export inhibitor leptomycin B. Expression of exogenous E-cadherin down-regulated nuclear p120(ctn) whereas activation of protein kinase C increased the level of nuclear p120(ctn). These results reveal molecular mechanisms controlling the subcellular distribution of p120(ctn).

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. British Journal of Pharmacology (1998) 123, 1655–1665; doi:10.1038/sj.bjp.0701766

Annals of the New York Academy of SciencesVolume 861, Issue 1 p. 236-237 Efficient Expression of Neurotransmitter Receptors in Mammalian Cells: Use of Inducible Expression Systems P. VANHOENACKER, Corresponding Author P. VANHOENACKER Department of Molecular Biology, University of Gent-VIB, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium Corresponding author: Tel: 32.9.264.5135; Fax: 32.9.264.5304; http://[email protected]Search for more papers by this authorI. VAN DEN BOGERD, I. VAN DEN BOGERD Department of Molecular Biology, University of Gent-VIB, K. L. Ledeganckstraat 35, B-9000 Gent, BelgiumSearch for more papers by this authorW. GOMMEREN, W. GOMMEREN Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, BelgiumSearch for more papers by this authorW.H.M. L. LUYTEN, W.H.M. L. LUYTEN Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, BelgiumSearch for more papers by this authorJ. E. LEYSEN, J. E. LEYSEN Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, BelgiumSearch for more papers by this authorG. HAEGEMAN, G. HAEGEMAN Department of Molecular Biology, University of Gent-VIB, K. L. Ledeganckstraat 35, B-9000 Gent, BelgiumSearch for more papers by this author P. VANHOENACKER, Corresponding Author P. VANHOENACKER Department of Molecular Biology, University of Gent-VIB, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium Corresponding author: Tel: 32.9.264.5135; Fax: 32.9.264.5304; http://[email protected]Search for more papers by this authorI. VAN DEN BOGERD, I. VAN DEN BOGERD Department of Molecular Biology, University of Gent-VIB, K. L. Ledeganckstraat 35, B-9000 Gent, BelgiumSearch for more papers by this authorW. GOMMEREN, W. GOMMEREN Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, BelgiumSearch for more papers by this authorW.H.M. L. LUYTEN, W.H.M. L. LUYTEN Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, BelgiumSearch for more papers by this authorJ. E. LEYSEN, J. E. LEYSEN Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, BelgiumSearch for more papers by this authorG. HAEGEMAN, G. HAEGEMAN Department of Molecular Biology, University of Gent-VIB, K. L. Ledeganckstraat 35, B-9000 Gent, BelgiumSearch for more papers by this author First published: 07 February 2006 https://doi.org/10.1111/j.1749-6632.1998.tb10197.xCitations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume861, Issue1ADVANCES IN SEROTONIN RECEPTOR RESEARCH: MOLECULAR BIOLOGY, SIGNAL TRANSDUCTION, AND THERAPEUTICSa December 1998Pages 236-237 RelatedInformation

Heterologous expression of cloned receptor subtypes for screening programs has become a real necessity for a modern pharmaceutical company. As the expression levels obtained so far are often low or unstable, we addressed this problem by using an inducible promoter system, i.e. the interferon-inducible mouse Mxl promoter. Using the gene coding for chloramphenicol acetyltransferase (CAT) as a reporter gene, we tested the inducibility of this promoter in the murine cell line L929, We found that background expression was low and that a distinct interferon-induced expression could be obtained. CAT expression reached its maximum at approximately 15 ng CAT/mg protein after induction for 24 hr with 1000 U/ml murine interferon-beta; the induction ratio was 150-fold. Next, L929 cells were transfected with four different human serotonin (5HT) receptor cDNAs (5HT(1A), 5HT(2A), 5HT(1D beta) and 5HT(1E)) under the control of the same Mxl promoter fragment. Also in this case well-regulated serotonin receptor-expressing clones were isolated. B-max values varied from 3100 fmol/mg protein for the 5HT(2A) receptor, 3300 fmol/mg protein for the 5HT(1D beta) receptor, 9800 fmol/mg protein for the 5HT(1E) receptor, and even up to 10400 fmol/mg protein for the 5HT(1A) receptor. Furthermore, the expression levels were shown to remain stable during serial propagation for at least one year, demonstrating the usefulness of this expression system. In fact, the 5HT(1D beta) receptor-expressing cells were used in the characterization of a new antimigraine agent, viz, alniditan.

Alniditan is a new migraine-abortive agent. It is a benzopyran derivative and therefore structurally unrelated to sumatriptan and other indole-derivatives and to ergoline derivatives. The action of sumatriptan is thought to be mediated by 5-hydroxytryptamine (5-HT)1D-type receptors. We investigated the receptor-binding profile in vitro of alniditan compared with sumatriptan and dihydroergotamine for 28 neurotransmitter receptor subtypes, several receptors for peptides and lipid-derived factors, ion channel-binding sites, and monoamine transporters. Alniditan revealed nanomolar affinity for calf substantia nigra 5-HT1D and for cloned h5-HT1D alpha, h5-HT1D beta and h5-HT1A receptors (Ki = 0.8, 0.4, 1.1, and 3.8 nM, respectively). Alniditan was more potent than sumatriptan at 5-HT1D-type and 5-HT1A receptors. Alniditan showed moderate-to-low or no affinity for other investigated receptors; sumatriptan showed additional binding to 5-HT1F receptors. Dihydroergotamine had a much broader profile with high affinity for several 5-HT, adrenergic and dopaminergic receptors. In signal transduction assays using cells expressing recombinant h5-HT1D alpha, h5-HT1D beta, or h5-HT1A receptors, alniditan (like 5-HT) was a full agonist for inhibition of stimulated adenylyl cyclase (IC50 = 1.1, 1.3, and 74 nM, respectively, for alniditan). Therefore, in functional assays, the potency of alniditan was much higher at 5-HT1D receptors than at 5-HT1A receptors. We further compared the properties of [3H]alniditan, as a new radioligand for 5-HT1D-type receptors, with those of [3H]5-HT in membrane preparations of calf substantia nigra, C6 glioma cells expressing h5-HT1D alpha, and L929 cells expressing h5-HT1D beta receptors. [3H]Alniditan revealed very rapid association and dissociation binding kinetics and showed slightly higher affinity (Kd = 1-2 nM) than [3H]5-HT. We investigated 25 compounds for inhibition of [3H]alniditan and [3H]5-HT binding in the three membrane preparations; Ki values of the radioligands were largely similar, although some subtle differences appeared. Most compounds did not differentiate between 5-HT1D alpha and 5-HT1D beta receptors, except methysergide, ritanserin, ocaperidone, risperidone, and ketanserin, which showed 10-60-fold higher affinity for the 5-HT1D alpha receptor. The Ki values of the compounds obtained with 5-HT1D receptors in calf substantia nigra indicated that these receptors are of the 5-HT1D beta-type. We demonstrated that alniditan is a potent agonist at h5-HT1D alpha and h5-HT1D beta receptors; its properties probably underlie its cranial vasoconstrictive and antimigraine properties.

In a number of eukaryotic cell types, the promoter of the interleukin-6 (IL-6) gene is inducible by several agents. We studied this inducibility in two human (MG63 and HeLa H21) and four simian cell lines (BSC-1, AP8, CV-1 and VERO). Furthermore, we also investigated whether this inducibility was maintained when a heterologous gene was placed under control of the human IL-6 promoter. As a heterologous gene we used the Simian Virus 40 (SV40) large T antigen, because of its stimulatory effects on SV40-based expression vectors. For the human cell lines tested, we observed that this gene was equally well induced and controlled as the endogenous IL-6 gene. In contrast, the aforementioned simian cell lines were only slightly inducible for IL-6 production; moreover, after transfection with the IL-6 promoter--SV40 T antigen gene construct, a continuous, low level of expression was found, even in those lines which only showed a low (CV-1) or no (BSC-1) endogenous IL-6 background. This apparent difference between the human and simian cells analyzed does not reflect a species specificity, but presumably is related to a different differentiation state of these cells. Furthermore, the IL-6 gene induction could be correlated with activation of the required transcription factor NF-kappa B.