The serotonin 5-HT7 receptors: two decades of research.

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.

Biochemical and pharmacological study of N-linked glycosylation of the human serotonin 5-HT7a receptor.

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-HT(7) receptor. Western blot analysis of HEK293T cells transiently expressing the 5-HT(7(a)) receptor in the presence of tunicamycin gave rise to a band shift, indicating the existence of an N-glycosylated form of the 5-HT(7(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 [(3) H]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-HT(7) receptor.

Cloning, genomic organization and functionality of 5-HT(7) 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.

Influence of the antipsychotic drug pipamperone on the expression of the dopamine D4 receptor.

The dopamine D4 receptor is a G protein-coupled receptor that binds with high affinity various antipsychotics. The receptor may be involved in attention/cognition, and in genetic studies a polymorphic repeat sequence in its coding sequence has been associated with attention deficit/hyperactivity disorder. We developed an inducible episomal expression system based on the reverse tetracycline transactivator and Epstein-Barr viral sequences. In HEK293rtTA cells expressing the dopamine D4 receptor from this episomal expression vector, addition of doxycycline in combination with sodium butyrate and trichostatin A induces high levels of receptor expression, resulting in 1970 +/- 20 fmol/mg membrane protein. Addition of the dopamine D4 receptor and serotonin 5-HT2A receptor antagonist pipamperone to these cells further increased the expression of the dopamine receptor, reaching 3800 +/- 60 fmol/mg membrane protein. This up-regulation was not restricted to the dopamine D4 receptor but was also found for the serotonin 5-HT2A receptor. We further provide evidence that the increase in receptor expression is not due to increased mRNA synthesis. As pipamperone could rescue the expression of a folding mutant of the dopamine D4 receptor (M345), we propose that pipamperone acts as a pharmacological chaperone for correct receptor folding thereby resulting in an increased dopamine D4 receptor expression. This study describes a strong and inducible expression system for proteins, difficult to express in other heterologous expression systems. This study also demonstrates that pipamperone, an antipsychotic, acts as a pharmacological chaperone and by doing so, increases the expression level of the dopamine D4 receptor. The fact that ligands can also act as pharmacological chaperones is a fairly new additional element in the regulation of receptor expression levels with potential great impact in drug treatment.