Key Determinants of Nucleotide-Activated G Protein-Coupled P2Y₂ Receptor Function Revealed by Chemical and Pharmacological Experiments, Mutagenesis and Homology Modeling

Abstract

The P2Y(2) receptor, which is activated by UTP, ATP, and dinucleotides, was studied as a prototypical nucleotide-activated GPCR. A combination of receptor mutagenesis, determination of its effects on potency and efficacy of agonists and antagonists, homology modeling, and chemical experiments was applied. R272 (extracellular loop EL3) was found to play a gatekeeper role, presumably responsible for recognition and orientation of the nucleotides. R272 is also directly involved in binding of dinucleotides, which behaved as partial agonists. Y118A (3.37) mutation led to dramatically reduced efficacy of agonists; it is part of the entry channel as well as the triphosphate binding site. While the Y114A (3.33) mutation did not have any effect on agonist activities, the antagonist Reactive Blue 2 (6) was completely inactive at that mutant. The disulfide bridge Cys25-Cys278 was found to be important for agonist potency but neither for agonist efficacy nor for antagonist potency.

Synthesis, characterization, and in vitro evaluation of the selective P2Y2 receptor antagonist AR-C118925

“…The newly formed precipitate was filtered off and washed with cold water (2 × 2 ml). 6 , and P2Y 11 , rat P2Y 6 , and P2X receptor subtypes Transfection was performed as previously described [37]. Briefly, the coding sequence of the respective receptor was cloned into the pQCXIN or pLXSN retroviral vector, amplified, purified, and sequenced prior to the transfection of GP + envAM-12 packaging cells together with vesicular stomatitis virus G (VSV-G) protein DNA using lipofectamine 2000.…”

“…The calcium measurements for the P2Y 11 and P2X receptors have also been described before [37]. In summary, 1321N1 human astrocytoma cells stably transfected with the coding sequence for the respective receptor were harvested with 0.05 % trypsin/0.6 mM EDTA and rinsed with culture medium.…”

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

“…We have now experimentally supported the assumption that probably no disulfide bond is formed, by measuring ligand binding and receptor activation (by cAMP accumulation studies) without and after preincubation with the disulfide-reducing agent DTT. In contrast to many other GPCRs that require an intact extracellular disulfide bond for proper functioning [16,48,52], the rAdeR did not show any difference, neither in adenine binding, nor in adenine-induced adenylate cyclase inhibition (Fig. 10) after incubation with DTT to reduce accessible disulfide bonds.…”

“…2) [45][46][47]; it also showed somewhat reduced affinity and potency. In contrast, several of the mutants displayed increased potency of adenine up to 14 [48,49].…”

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

“…2+ release experiments were performed as previously described [27]: Transfected cells of two 175-cm 2 flask were detached, incubated for 30 min at normal culture conditions, and subsequently spun down at 200×g for 5 min. Pelleted cells were then resuspended in 1 ml of Krebs-HEPES buffer.…”