1 We have previously shown that, in glioma C6 cells, two nucleotide ADP-sensitive receptors coexist: P2Y 1 , coupled to PLC and responsible for Ca 2 þ release, and P2Y 12 , negatively coupled to adenylate cyclase. In the present study, we examined the effects of the stimulation of these two receptors on ERK1/2 and PI3-K activation, and cell proliferation in either serum-deprived or nonstarved C6 cells. 2 In response to ADP and its analogues, in serum-starved cells, both p44 ERK1 and p42 ERK2 were activated in a time-dependent manner, as monitored by Western blot analysis using an antiphosphop42/p44 MAPK antibody. The phosphorylation was reduced both by removal of the extracellular Ca 2 þ and partially or almost completely by MRS2179 or AR-C69931MX, specific antagonists of the P2Y 1 and P2Y 12 receptors, respectively. The inhibitory effect of antagonists was additive. These data indicate the involvement of both receptors, P2Y 1 and P2Y 12 , in the ERK1/2 activation, but the P2Y 12 receptor contribution predominates. 3 ERK1/2 activity was positively correlated with cell proliferation of cultured glioma C6 cells. 4 In nonstarved cells, ADP markedly decreased the PI3-K activity. In contrast, in serum-starved cells, ADP evoked an increase in the PI3-K activity. Blocking of the P2Y 1 receptor by MRS2179 additionally increased this ADP response. These results suggest that the P2Y 1 receptor has an inhibitory and the P2Y 12 receptor a stimulatory effect on PI3-K signalling pathway. 5 RT-PCR analysis revealed different mRNA expression of both receptors in starved and nonstarved cells. In nonstarved cells, the P2Y 1 receptor mRNA predominates, whereas in serumdeprived cells the expression of P2Y 12 mRNA becomes more pronounced.

] i . 6 ADP inhibited isoproterenol-induced cyclic AMP accumulation. PTX blocked this eect, but PPADS did not. 7 RT ± PCR analysis revealed the molecular identity of P2Y receptors expressed by glioma C6 cells to be both P2Y 1 and P2Y 2 . 8 It is concluded that both P2Y 1 and P2Y 2 receptors co-exist in glioma C6 cells. ADP acts as agonist of the ®rst, and ATP and UTP of the second one. Both receptors are linked to phospholipase C (PLC).

In this study we characterized the subtypes of nucleotide P2Y receptors that respond to ADP in glioma C6 cells. Direct visualization of phosphatidylinositol 4,5-bisphosphate at the cell surface revealed that extracellular ADP activates phospholipase C (PLC). Knock-down of P2Y 1 receptor with antisense oligonucleotide, as well as treatment with MRS2179 and pyridoxal-phosphate-6-azophenyl-2P P,4P P-disulfonic acid (P2Y 1 antagonists), attenuates receptor-mediated PLC activity. Adenylyl cyclase inhibition by ADP remains unchanged under these conditions. Reverse transcription-PCR analysis showed that P2Y 12 receptor is expressed in C6 cells. We therefore conclude that, in glioma C6 cells, two P2Y receptor subtypes are present: P2Y 1 , coupled to PLC, and P2Y 12 , negatively coupled to adenylyl cyclase. ß

†These authors contributed equally to this work.Regulator of ubiquitous kinase/Cbl-interacting protein of 85 kDa (Ruk/CIN85) and CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS) comprise a family of vertebrate adaptor proteins involved in several important cellular processes, including downregulation of activated receptor tyrosine kinases, regulation of cytoskeletal rearrangements, phosphatidylinositol 3-kinase (PI 3-kinase) signalling and apoptosis. The role of Ruk/CIN85 as a scaffold protein involved in membrane trafficking processes has been demonstrated in model cell systems. However, intracellular localization of endogenous Ruk/CIN85 has never been comprehensively assessed. We carried out detailed studies of subcellular distribution of Ruk/CIN85 in adherent cultured human cells using antibodies that recognize distinct epitopes of the protein and revealed a punctate immunostaining pattern, common for proteins involved in intracellular trafficking processes. Our data indicate that Ruk/CIN85 is distributed between several different membrane trafficking compartments, but the major pool of Ruk/CIN85 is associated with the Golgi complex, mainly with a subpopulation of COPI-coated vesicles involved in retrograde endoplasmic reticulum-Golgi and intra-Golgi transport. This localization pattern is dependent on the integrity of Golgi complex and intact microtubular network. Only a small pool of Ruk/CIN85 is present in compartments involved in clathrin-mediated endocytosis and sorting. These results suggest that endogenous Ruk/CIN85 may be involved in regulation of specific membrane trafficking processes.