To assess the role of G16, a trimeric G protein exclusively expressed in hematopoietic cells, Galpha16 antisense RNA was stably expressed in human erythroleukemia (HEL) cells. Western blot analysis showed that in transfected cell lines, the expression of endogenous Galpha16 protein was suppressed, but the expression of Galphaq/11, Galphai2, and Galphai3 remained unaffected. Suppression of Galpha16 in transfected HEL cells did not interfere with transient elevations of intracellular free Ca2+ concentrations induced by prostaglandin E1 (PGE1), 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 microM, respectively, apparently by stimulating P2U purinoceptors. By contrast, Ca2+ mobilization by UTP or ATP was completely abrogated in Galpha16-suppressed cells, indicating specific coupling of G16 to P2U purinoceptors. Pertussis toxin inhibited the effect of UTP in parental HEL cells by 57.6 +/- 4.9%. These data indicate that signaling by the P2U purinoceptor obligatorily requires G16 but may be modulated further by activation of Gi. Priming of HEL cells with UTP or ATP prior to stimulation with PGE1 markedly enhanced the PGE1-induced intracellular Ca2+ release. This indirect, potentiating effect of UTP and ATP was not impaired in Galpha16-suppressed cells but was inhibited by pertussis toxin, indicating that functional P2U purinoceptors are present on these cells and that the potentiating effect primarily depends on Gi. The data demonstrate (i) that Galpha16 antisense RNA selectively inhibits endogenous Galpha16 protein expression in HEL cells; (ii) that stimulation of endogenous P2U (P2Y2) purinoceptors leads to the mobilization of intracellular Ca2+ by a mechanism that strictly depends on Galpha16; and (iii) that P2U purinoceptors in HEL cells can communicate with two distinct signaling pathways diverging at the G protein level.