Characterization of new G protein-coupled adenine receptors in mouse and hamster

Abstract

The nucleobase adenine has previously been reported to activate G protein-coupled receptors in rat and mouse. Adenine receptors (AdeR) thus constitute a new family of purine receptors, for which the designation "P0-receptors" has been suggested. We now describe the cloning and characterization of two new members of the AdeR family from mouse (MrgA10, termed mAde1R) and hamster (cAdeR). Both receptors were expressed in Sf9 insect cells, and radioligand binding studies were performed using [ 3 H] adenine. Specific binding of the radioligand was detected in transfected, but not in untransfected cells, and K D values of 286 nM (mAde1R, B max 1.18 pmol/mg protein) and 301 nM (cAdeR, B max 17.7 pmol/mg protein), respectively, were determined. A series of adenine derivatives was investigated in competition binding assays. Minor structural modifications generally led to a reduction or loss of affinity, with one exception: 2-fluoroadenine was at least as potent as adenine itself at the cAdeR. Structure-activity relationships at all AdeR orthologs and subtypes investigated so far were similar, but not identical. For functional analyses, the cAdeR was homologously expressed in Chinese hamster ovary (CHO) cells, while the mAde1R was heterologously expressed in 1321N1 astrocytoma cells. Like the previously described AdeRs from rat (rAdeR) and mouse (mAde2R), the mAde1R (EC 50 9.77 nM) and the cAdeR (EC 50 51.6 nM) were coupled to inhibition of adenylate cyclase. In addition, the cAdeR from hamster expressed in CHO cells produced an increase in intracellular calcium concentrations (EC 50 6.24 nM) and was found to be additionally coupled to G q proteins.

Mas and Its Related G Protein–Coupled Receptors, Mrgprs

“…In mouse, two members of the MrgprA subfamily, mMrgprA9 and A10, have also been described to bind adenine, albeit with somehow lower affinities (K d : 113 and 286 nM, respectively), and to inhibit cAMP generation (von Kügelgen et al, 2008;Knospe et al, 2013;Thimm et al, 2013). The same group has discovered an adenine binding homolog in Chinese hamster, but there has not yet been any adenine receptor of the Mrgpr family found in humans.…”

Purinergic neurone-glia signalling in cognitive-related pathologies

“…After being released, ATP is rapidly degraded by numerous ectonucleotidases (Zimmermann et al, 2012) thus generating a trail of ADP, AMP and adenosine, which also act as signalling molecules. At the cellular level effects of ATP are mediated through families of purinoceptors broadly classified as metabotropic adenine P0 receptors, metabotropic P1 adenosine receptors, metabotropic P2Y purinoceptors and ionotropic P2X purinoceptors Thimm et al, 2013;Verkhratsky and Burnstock, 2014). The latter 3 types of the receptors are widely expressed in all types of glial cells ; glial expression of P0 receptors has not yet been studied.…”

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].…”