The P2U Purinoceptor Obligatorily Engages the Heterotrimeric G Protein G16 to Mobilize Intracellular Ca2+ in Human Erythroleukemia Cells

Abstract

To assess the role of G 16 , a trimeric G protein exclusively expressed in hematopoietic cells, G ␣16 antisense RNA was stably expressed in human erythroleukemia (HEL) cells. Western blot analysis showed that in transfected cell lines, the expression of endogenous G ␣16 protein was suppressed, but the expression of G ␣q/11 , G ␣i2 , and G ␣i3 remained unaffected. Suppression of G ␣16 in transfected HEL cells did not interfere with transient elevations of intracellular free Ca 2؉ concentrations induced by prostaglandin E 1 (PGE 1 ), platelet-activating factor, or thrombin. In parental HEL cells, UTP and ATP mobilized Ca 2؉ from intracellular stores with half-maximum effective concentrations of 3.6 ؎ 0.7 and 4.7 ؎ 1.6 M, respectively, apparently by stimulating P 2U purinoceptors. By contrast, Ca 2؉ mobilization by UTP or ATP was completely abrogated in G ␣16 -suppressed cells, indicating specific coupling of G 16 to P 2U purinoceptors. Pertussis toxin inhibited the effect of UTP in parental HEL cells by 57.6 ؎ 4.9%. These data indicate that signaling by the P 2U purinoceptor obligatorily requires G 16 but may be modulated further by activation of G i . Priming of HEL cells with UTP or ATP prior to stimulation with PGE 1 markedly enhanced the PGE 1 -induced intracellular Ca 2؉ release. This indirect, potentiating effect of UTP and ATP was not impaired in G ␣16 -suppressed cells but was inhibited by pertussis toxin, indicating that functional P 2U purinoceptors are present on these cells and that the potentiating effect primarily depends on G i . The data demonstrate (i) that G ␣16 antisense RNA selectively inhibits endogenous G ␣16 protein expression in HEL cells; (ii) that stimulation of endogenous P 2U (P2Y 2 ) purinoceptors leads to the mobilization of intracellular Ca 2؉ by a mechanism that strictly depends on G ␣16 ; and (iii) that P 2U purinoceptors in HEL cells can communicate with two distinct signaling pathways diverging at the G protein level.

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

“…Purity (NMR), 66 %; main impurities, 24 % furan-2-carboxylic acid ethyl ester and 10 % ethylbenzene (both imported from the previous step). 1 H-NMR (500 MHz, CDCl 3 ) δ (ppm) = 1.34 (t, J = 7.3 Hz, 3H), 2.36 (s, 3H, CH 3 ), 4.32 (q, J = 7.3 Hz, 2H), 6.09 (dq, J = 0.9 Hz, J = 3.5 Hz, 1H), and 7.05 (dd, J = 0.5 Hz, J = 3.5 Hz, 1H). 5-(Bromomethyl)-furan-2-carboxylic acid ethyl ester (8) 5-Methylfuran-2-carboxylic acid ethyl ester (2.82 g, 12.8 mmol (considering the degree of purity)), N-bromosuccinimide (2.49 g, 14 mmol), and AIBN (0.1 g) were suspended in tetrachloromethane (30 ml) and stirred at 70°C for 2 h. After cooling to rt, the solid was filtered off and washed with tetrachloromethane (5 ml).…”

“…Purity (NMR), 68 mol% (78 mass%), a single impurity was present: 32 mol% (22 mass%) furan-2-carboxylic acid ethyl ester. 1 H-NMR (500 MHz, CDCl 3 ) δ (ppm) = 1.36 (t, J = 7.3 Hz, 3H), 4.35 (q, J = 7.3 Hz, 2H), 4.47 (s, 2H), 6.47 (d, J = 3.5 Hz, 1H), and 7.09 (d, J = 3.6 Hz, 1H).…”

“…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.…”

“…These are the P2Y 1 -like receptors P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 , as well as the P2Y 12 -like subtypes P2Y 12 , P2Y 13 , and P2Y 14 [1]. The numbers missing in this sequence correspond to nonmammalian orthologs or to receptors that have structural similarities but do not respond to nucleotides.…”

“…As a result, the second messengers inositol trisphosphate (IP 3 ) and diacylglycerol are produced that mediate the release of calcium ions from intracellular stores, and protein kinase C activation, respectively. In addition, the P2Y 2 receptor appears to couple to other G proteins as well, including G o [4], G 12 [5], and G 16 [6]. P2Y 2 receptor mRNA is expressed in a range of different human organs and tissues, including skeletal muscle and heart, at more moderate levels in the spleen, intestine, immune cells, bone marrow, and lungs, as well as to a lower extent also in kidneys, liver, stomach, pancreas, bones, and different regions of the brain [7].…”