Combined in situ hybridisation, northern blot analysis, and receptor binding studies in clones expressing different levels of the human 5-HT1A receptor.

In order to set up the technique of semi-quantitative in situ hybridisation to detect the serotonin receptor mRNA levels in brain tissue, a panel of three Swiss 3T3 cell clones (named clones 66, 53 and 47) expressing the human 5-HT1A receptor at different densities were used as a model. The clones were generated by limiting dilution from pools of stably transfected cells. In addition membranes were prepared from each clone to perform receptor binding studies. Clones 66, 53, and 47 showed saturable binding for the agonist [3H]-8-OH-DPAT, with receptor densities (Bmax) of 227 +/- 86, 548 +/- 107 and 1505 +/- 212 fmol/mg protein respectively, and with corresponding affinity constants (pKd) of 8.8 +/- 0.1, 9.1 +/- 0.1, and 9.1 +/- 0.1 nM, respectively. Northern blot analysis using a specific probe for the 5-HT1A receptor revealed the presence of a single 1.56 kilobase mRNA species in the 5-HT1A receptor clones but not in control cells. In situ hybridisation studies were performed by measuring the 5-HT1A receptor mRNA levels in these three 5-HT1A transfectants using [35S]alphaCTP labeled riboprobes (sense and anti-sense). The following rank order of receptor mRNA expression was found for clones 66, 53 and 47 respectively: 0.140 +/- 0.001, 0.365 +/- 0.045 and 0.835 +/- 0.115 (relative optical density units). With the sense probe no specific labelling was observed. In conclusion, a positive correlation was found between receptor density (Bmax) and receptor mRNA expression (semi-quantitative in situ hybridisation) using human 5-HT1A receptor clones with different expression levels.

Genomic organisation and functional expression of the gene encoding the human serotonin 5-HT2C receptor.

The 5-HT2C receptor gene is unique among the members of the 5-HT receptor family by virtue of its genomic organisation. The human 5-HT2C receptor gene, unlike many other genes for guanine nucleotide binding (G)-proteins, contains three introns which interrupt the coding sequence into four exons. The first two introns are at equivalent positions as compared to the intervening sequences previously found in the 5-HT2(A) receptor gene, suggesting a close evolutionary relationship between both genes. Southern blot analysis shows that the 5-HT2C receptor gene is a single copy gene. Furthermore, we report the functional expression of a complementary DNA for the 5-HT2C receptor, cloned from hippocampal RNA. Membranes prepared from NIH 3T3 cells stably expressing the 5-HT2C receptor cDNA, displayed a single population of high affinity sites for the antagonist [3H]mesulergine (Kd = 2.9 +/- 0.4 nM, Bmax = 44.3 +/- 7.2 pmol/mg protein) as well as for [3H]5-HT (Kd = 9.9 +/- 0.7 nM, Bmax = 13.6 +/- 1.0 pmol/mg protein). Displacement of [3H]mesulergine and [3H]5HT binding by ligands indicated a pharmacological similarity of these binding sites with porcine and rat choroid plexus 5-HT2C receptors. Furthermore, activation of the 5-HT2C receptor with 5-HT results in an increased phospholipase C activity.

Genomic organization, coding sequence and functional expression of human 5-HT2 and 5-HT1A receptor genes.

The family of serotonin receptors consists of at least eight distinct subtypes, divided into four classes based on their pharmacological and functional characteristics. Here we report the cloning and expression in Swiss 3T3 cells of the human 5-HT2 and 5-HT1A receptor subtypes. Both genes encode functional receptors for 5-HT, that differ considerably in genomic structure, primary amino acid sequence, pharmacology and signal transduction. The 5-HT1A receptor transfectants displayed a single high affinity site for the agonist [3H](+/-)-8-hydroxy-2-(di-n-propylamino)tetralin HBr ([3H]8-OH-DPAT) and a pharmacological profile specific for the 5-HT1A receptor. In these transfectants, 5-HT mediated a dose-dependent inhibition of forskolin-stimulated cAMP levels. Cells expressing the 5-HT2 receptor exhibited high affinity binding for the antagonist [3H]ketanserin with a 5-HT2 receptor specific pharmacological profile. In these cells 5-HT activated phospholipase C in a dose-dependent manner. The 5-HT2 receptor displayed a genomic organization quite different from the 5-HT1A, 5-HT1B and 5-HT1D receptor subtypes. While these receptors are encoded by one single exon, the 5-HT2 receptor is encoded by three exons separated by two introns. The latter finding adds and additional molecular criterion for receptor classification.