Characterization of an orphan G protein-coupled receptor localized in the dorsal root ganglia reveals adenine as a signaling molecule

Abstract

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...

Nucleotides and nucleoside signaling in the regulation of the epithelium to mesenchymal transition (EMT)

“…In addition, ADORA1 activation can directly activate K + channels and inhibit Q-, P-and N-type Ca 2+ channels [24]. The proposed P0 receptors are also GPCR, the first one was cloned in 2002 from rat tissue [25], recently has been cloned from, mouse and guinea pig [26,27], P0 receptor are mainly coupled to Gi protein and in consequence to the inhibition of adenylate cyclase [18,25,26], and are expressed in lung, ovaries, kidneys, and nervous system [25].…”

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

“…They are subdivided into two families, the adenosine-activated P1 receptors and the nucleotide-activated P2 receptors [1]. The nucleobase adenine has recently been found to be a signaling molecule as well and was identified as the endogenous ligand of the Mas-related gene receptor A (MrgA) from rat, now designated rat adenine receptor (rAdeR) [2]. Meanwhile, two mouse AdeRs (mAde1R and mAde2R) have been identified and characterized, which are activated by adenine in nanomolar concentrations [3].…”

“…AdeRs belong to the rhodopsin-like class A G protein-coupled receptor (GPCR) family. They have been shown to be G i protein-coupled, leading to the inhibition of adenylate cyclase [2,3,5,6]. 1 Additional coupling to G q proteins has recently been demonstrated for the hamster AdeR (cAdeR) when homologously expressed in Chinese hamster ovary (CHO) cells.…”

“…1 Additional coupling to G q proteins has recently been demonstrated for the hamster AdeR (cAdeR) when homologously expressed in Chinese hamster ovary (CHO) cells. 1 The rat AdeR is highly expressed in small diameter neurons of dorsal root ganglia and can also be detected in the brain cortex, hypothalamus, lung, ovaries, kidneys, and small intestine [2]. Adenine applied to the spinal cord of rats exhibited proalgesic effects [7].…”

“…In patients with chronic renal failure, elevated adenine concentrations were measured, which increased with the progression of the disease [14], indicating a potential role for adenine as a signaling molecule in the human kidney as well. However, sequence analyses have not led to the identification of a direct human ortholog of the rAdeR [2]. Although human GPCR(s) for adenine appear to exist, their molecular identity cannot be easily identified by a bioinformatics approach.…”