The cloning of novel G protein-coupled receptors and the search for their natural ligands, a process called reverse pharmacology, is an excellent opportunity to discover novel hormones and neurotransmitters. Based on a degenerate primer approach we have cloned a G protein-coupled receptor whose mRNA expression profile indicates highest expression in the dorsal root ganglia, specifically in the subset of small neurons, suggesting a role in nociception. In addition, moderate expression was found in lung, hypothalamus, peripheral blood leukocytes, and ovaries. Guided by a receptoractivation bioassay, we identified adenine as the endogenous ligand, which activated the receptor potently and with high structural stringency. Therefore, we propose to name this receptor as the adenine receptor. Hormonal functions have already been demonstrated for adenine derivatives like 6-benzylaminopurine in plants and 1-methyladenine in lower animals. Here, we demonstrate that adenine functions as a signaling molecule in mammals. This finding adds a third family besides P1 and P2 receptors to the class of purinergic receptors. G protein-coupled receptors (GPCRs) have a superior success record as drug targets, which fueled the interest in the identification of novel GPCRs. As a consequence, reverse pharmacology (1), the process that leads from an orphan receptor to the identification of its endogenous ligand, already has yielded approximately 40 novel receptor͞ligand pairs (for a recent review see ref.2). In some cases, completely unknown hormones or neurotransmitters, such as nociceptin (3), prolactin-releasing peptide (4), apelin (5), and the orexins (6), were discovered.While cloning novel GPCRs by degenerate primer (PCR) we found a unique GPCR in a rat cortex cDNA preparation. Analysis of its sequence analysis by BLAST revealed that this receptor did not group within any of the GPCR families activated by a known ligand. The most closely related sequences-the sensory neuron-specific receptors (7) and the MAS-related gene (Mrg) receptors (8)-belong to families that contain only orphan receptors themselves. To characterize this additional receptor we mapped its tissue distribution and tried to identify its natural ligand. Materials and MethodsCloning and Expression of the Rat Adenine Receptor. The FastTrack 2.0 kit (Invitrogen) was used to isolate mRNA from rat brain cortex, which was then reverse-transcribed into cDNA with the SMART RACE (rapid amplification of cDNA ends) cDNA amplification kit (CLONTECH). The initial rat adenine receptor cDNA fragment was derived from a degenerate primer PCR containing primers complementary to the TM2 region (5Ј-AATCTGTTCCTGATGACGCTGGCGT-3Ј) and TM7 region (5Ј-GGTGGTTGAGGCAGCAATAGATGATGGGGTT-3Ј) (9). For the elongation of the PCR fragment to the full-length reading frame, the SMART RACE cDNA amplification kit was used. The full-length coding sequence (GenBank accession no. AJ311952) was subcloned into pcDNA3, and the resulting expression construct was used for transient and stable expression in mam...

Several variants of the serotonin 5-HT 4 receptor are known to be produced by alternative splicing. To survey the existence and usage of exons in humans, we cloned the human 5-HT 4 gene. Based on sequence analysis seven C-terminal variants (a-g) and one internal splice variant (h) were found. We concentrated in this study on the functional characterization of the novel splice variant h, which leads to the insertion of 14 amino acids into the second extracellular loop of the receptor. The h variant was cloned as a splice combination with the C-terminal b variant; therefore, we call this receptor 5-HT 4(hb) . This novel receptor variant was expressed transiently in COS-7 cells, and its pharmacological profile was compared with those of the previously cloned 5-HT 4(a) and 5-HT 4(b) isoforms, with the latter being the primary reference for the h variant. In competition binding experiments using reference 5-HT 4 ligands, no significant differences were detected. However, the broadly used 5-HT 4 antagonist GR113808 discriminated functionally among the receptor variants investigated. As expected, it was an antagonist on the 5-HT 4(a) and 5-HT 4(b) variant but showed partial agonistic activity on the 5-HT 4(hb) variant. These data emphasize the importance of variations introduced by splicing for receptor pharmacology and may help in the understanding of conflicting results seen with 5-HT 4 ligands in different model systems. Key Words: Serotonin 5-HT 4 receptor-Serotonin 5-HT 4(hb) variantSplice variant-G protein-coupled receptor-Gene structure -Human-Serotonin.