P-2Y purinoceptor subtypes recruit different Mek activators in astrocytes

1 Extracellular ATP can function as a glial trophic factor as well as a neuronal transmitter. In astrocytes, mitogenic signalling by ATP is mediated by metabotropic P-2Y receptors that are linked to the extracellular signal regulated protein kinase (Erk) cascade, but the types of P-2Y receptors expressed in astrocytes have not been defined and it is not known whether all P-2Y receptor subtypes are coupled to Erk by identical or distinct signalling pathways. 2 We found that the P-2Y receptor agonists ATP, ADP: UTP and 2-methylthioATP (2MeSATP) activated Erk and its upstream activator MAP/Erk kinase (Mek). cRaf-1, the first kinase in the Erk cascade, was activated by 2MeSATP, ADP and UTP but, surprisingly, cRaf-1 was not stimulated by ATP. Furthermore, ATP did not activate B-Raf, the major isoform of Raf in the brain, nor other Mek activators such as Mek kinase 1 (MekK1) and MekK2/3. 3 Reverse transcriptase-polymerase chain reaction (RT-PCR) studies using primer pairs for cloned rat P-2Y receptors revealed that rat cortical astrocytes express P-2Y1, a receptor subtype stimulated by ATP and ADP and their 2MeS analogues, as well as P-2Y2 and P-2Y4, subtypes in rats for which ATP and UTP are equipotent. Transcripts for P-2Y6, a pyrimidine-preferring receptor, were not detected. 4 ATP did not increase cyclic AMP levels, suggesting that P-2Y11, an ATP-preferring receptor, is not expressed or is not linked to adenylyl cyclase in rat cortical astrocytes. 5 These signal transduction and RT-PCR experiments reveal differences in the activation of cRaf-1 by P-2Y receptor agonists that are inconsistent with properties of the P-2Y1, P-2Y2 and P-2Y4 receptors shown to be expressed in astrocytes, i.e. ATP not equal UTP; ATP not equal 2MeSATP, ADP. This suggests that the properties of the native P-2Y receptors coupled to the Erk cascade differ from the recombinant P-2Y receptors or that astrocytes express novel purine-preferring and pyrimidine-preferring receptors coupled to the ERK cascade.