The P-2U purinoceptor obligatorily engages the heterotrimeric G protein G(16) to mobilize intracellular Ca2+ in human erythroleukemia cells

To assess the role of G(16), a trimeric G protein exclusively expressed in hematopoietic cells, G(alpha 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(alpha 16) protein was suppressed, but the expression of G(alpha q/11), G(alpha i2), and G(alpha i3) remained unaffected, Suppression of G(alpha 16) in transfected HEL cells did not interfere with transient elevations of intracellular free Ca2+ concentrations induced by prostaglandin E(1) (PGE(1)), platelet-activating factor, or thrombin, In parental HEL cells, UTP and ATP mobilized Ca2+ from intracellular stores with half-maximum effective concentrations of 3.6 +/- 0.7 and 4.7 +/- 1.6 mu M, respectively, apparently by stimulating P-2U purinoceptors. By contrast, Ca2+ mobilization by UTP or ATP was completely abrogated in G(alpha 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 Ca2+ release. This indirect, potentiating effect of UTP and ATP was not impaired in G(alpha 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(alpha 16) antisense RNA selectively inhibits endogenous G(alpha 16) protein expression in HEL cells; (ii) that stimulation of endogenous P-2U (P2Y(2)) purinoceptors leads to the mobilization of intracellular Ca2+ by a mechanism that strictly depends on G(alpha 16); and (iii) that P-2U purinoceptors in HEL cells can communicate with two distinct signaling pathways diverging at the G protein level.