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The 5-hydroxytryptamine (5-HT)7(a) receptor is a G-protein-coupled receptor critically involved in human psychiatric and neurological disorders. In the present study, we evaluate the presence and the functional role of N-glycosylation of the human 5-HT7 receptor. Western blot analysis of HEK293T cells transiently expressing the 5-HT7(a) receptor in the presence of tunicamycin gave rise to a band shift, indicating the existence of an N-glycosylated form of the 5-HT7(a) receptor. To further investigate this, we mutated the two predicted N-glycosylation sites (N5Q and N66Q) and compared the molecular mass of the immunoreactive bands with those of the wild-type receptor, indicating that both asparagines were N-glycosylated. The mutant receptors had the same binding affinity for [3H]5-CT and the same potency and efficacy with regard to 5-HT-induced activation of adenylyl cyclase. However, there was a reduction in maximal ligand binding for the single and double mutants compared to the wild-type receptor. Next, membrane labelling and immunocytochemical studies demonstrated that the N-glycosylation mutants were expressed at the cell surface. We conclude that N-glycosylation is not important for cell surface expression of the 5-HT7 receptor. [ABSTRACT FROM AUTHOR]

Dopamine D receptors (DRs) are G protein-coupled receptors that play a role in attention and cognition. In the present study, we investigated the dimerization properties of this receptor. Western blot analysis of the human DR, DR and DR revealed the presence of higher molecular weight immunoreactive bands, which might indicate the formation of receptor dimers and multimers. Homo- and heterodimerization of the receptors was confirmed by co-immunoprecipitation and bioluminescence resonance energy transfer studies. Although dimerization of a large number of G protein-coupled receptors has been described, the functional importance often remains to be elucidated. Folding efficiency is rate-limiting for DR biogenesis and quality control in the endoplasmic reticulum plays an important role for DR maturation. Co-immunoprecipitation and immunofluorescence microscopy studies using wild-type and a nonfunctional DR folding mutant show that oligomerization occurs in the endoplasmic reticulum and that this plays a role in the biogenesis and cell surface targeting of the DR. The different polymorphic repeat variants of the DR display differential sensitivity to the chaperone effect. In the present study, we show that this is also reflected by bioluminescence resonance energy transfer saturation assays, suggesting that the polymorphic repeat variants have different relative affinities to form homo- and heterodimers. In summary, we conclude that DRs form oligomers with different affinities and that dimerization plays a role in receptor biogenesis. We investigated the oligomerization properties of three polymorphic repeat variants of the hDR by immunoblot analysis, co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) studies. We demonstrate that hDR oligomerization occurs in the endoplasmic reticulum and plays a role in receptor biogenesis. Furthermore the number of repeats is important for the efficiency and affinity to form oligomers. [ABSTRACT FROM AUTHOR]

Abstract: The molecular mechanism regulating serotonin 5-HT7 receptor expression is still unclear. In this study we provide evidence that transcription of the 5-HT7 gene is at least partly regulated by Sp1 and Sp3. We isolated and sequenced >3000 bp of the upstream sequences and identified by RACE a number of transcriptional initiation sites over a region of 300 bp upstream of the coding region. This region has a high GC content, but contains no obvious TATA or a CAAT box. Besides a Sp1/Sp3 consensus motif, regulatory elements for AP2, Egr-1 and MAZ are also present. Transient transfection assays using deletion variants indicated that the GC-rich region is essential for full promoter activity. The role of Sp1 in this was confirmed by transient overexpression of both wild type Sp1 or dominant-negative forms. By gel shift and supershift analyses, targeting the Sp1 consensus sequence and the GC-rich region just upstream of the transcription initiation sites, binding of Sp1 and Sp3 was demonstrated. Both in vitro as well as in vivo experiments, using a cell line which endogenously expresses the 5-HT7 receptor, indicated that mithramycin A, an inhibitor of Sp1/3 transcription factor binding, was able to inhibit 5-HT7 promoter activity. Taken together these results support the essential role of Sp factors in regulating 5-HT7 promoter activity. [Copyright &y& Elsevier]

Abstract: The serotonin (5-HT) 5-HT7 receptors are expressed in both the central nervous system and in peripheral tissues. Receptor distribution studies and pharmacological studies have established that 5-HT7 receptors play an important role in the control of circadian rhythms and thermoregulation. Selective 5-HT7 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-HT7 receptor from mouse brain. These two new splice variants have almost identical sequences as the rat 5-HT7(b) and 5-HT7(c) splice variants and so were given the same name. Ligand binding assays ([3H]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. [Copyright &y& Elsevier]

Abstract: Three 5-HT7 receptor isoforms are expressed in rat and man, which differ in the amino acid sequence of their C-terminus. Thus far, no changes have been observed in the pharmacological profile of all three isoforms. To further elucidate the signal transduction pathway specific for these receptor variants, we screened for possible interacting proteins of the C-terminus of the h5-HT7(a) variant in a human foetal brain cDNA library. Using a yeast two-hybrid assay, we isolated PLAC-24/eIF3k as a possible interacting candidate. The association of PLAC-24 with all three receptor variants was observed and further reconfirmed in vivo by co-immunoprecipitation of PLAC-24 with the full-length receptor isoforms in transfected COS-7 cells. Studies with different deletion mutants of the receptor showed that the interaction between PLAC-24 and the receptor is not restricted to the C-terminus of the receptor. PLAC-24/eIF3k consists of 3 domains: an N-terminal HAM domain, a central WH domain and a C-terminal tail. We generated different domain constructs of PLAC-24, which indicated that the HAM and WH domain both interact with the 5-HT7(a) receptor. Overexpression of PLAC-24 in HEK293 cells, stably expressing the h5-HT7(a) receptor, caused a threefold augmentation in the expression levels of the receptor. Co-localisation studies in COS-7 cells showed that PLAC-24 relocates from the nucleus and perinuclear sites towards the plasma membrane upon co-expression with the receptor. On the other hand, the expression of domain variants of PLAC-24 seems to block the translocation of the receptor towards the membrane. These observations suggest that PLAC-24 may play a role in the transport and the stabilisation of newly synthesised 5-HT7 receptor towards the plasma membrane. [Copyright &y& Elsevier]

'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.