Adenosine nucleotides acting at the human P2Y(1) receptor stimulate mitogen-activated protein kinases and induce apoptosis

For the widely distributed P2Y receptors for nucleotides, the transductiona l and functional responses downstream of their coupling to G proteins are p oorly characterized. Here we describe apoptotic induction and the associate d differential stimulation of mitogen-activated protein (MAP) kinase family members by the human P2Y(1) receptor. The potent P2Y(1) receptor agonist, 2-methylthio-ADP (2-MeSADP), stimulated the extracellular-signal regulated kinases (ERK1/2) (EC50 similar to5 nM) as well as several, but not all isof orms detected, of the stress-activated protein kinase (SAPK) family. Phosph o-isoforms of p38 were unaffected. The induced kinase activity was blocked by the P2Y(1) receptor-selective antagonist, adenosine-2'-phosphate-5'-phos phate, but unaffected by pertussis toxin, In addition, the endogenous ligan d ADP, and significantly also 2-MeSATP, induced concentration-dependent pho sphorylation changes in the same MAP kinase family members. The sustained a ctivation of ERK1/2 was associated with Elk-1 phosphorylation that was abol ished by the MEK1 inhibitor, PD 98059, However, the concomitant transient a ctivation of the SAPKs was not sufficient to induce c-Jun or ATF-2 phosphor ylation, The transient phase of the ERK activity was partially inhibited ei ther by the phosphatidylinositol 3-kinase inhibitor, LY 294002, or the PKC inhibitor, Go 6976, In addition, the Src inhibitor, PP1, or expression of d ominant negative Has also attenuated the transient phase of ERK phosphoryla tion. In contrast, inhibition of Ras or Src had no effect on the sustained ERK activity, which was critically dependent on phosphatidylinositol 3-kina se, The transient SAPK activity was suppressed by expression of a dominant negative form of MKK4. Furthermore, this kinase-deficient mutant inhibited 2-MeSADP-induced caspase-3 stimulation and the associated decrease in cell number. In conclusion, adenosine di- and triphosphate stimulation of the hu man P2Y(1) receptor can transiently activate the Ras-ERK cascade via the co operative effects of phosphatidylinositol 3-kinase, Src and PKC, The sustai ned ERK stimulation, via a Ras-insensitive pathway, culminates in Elk-1 act ivation without inducing a proliferation effect. The transient SAPK activit y did not evoke transcription factor phosphorylation but was required for t he P2Y(1) receptor-mediated apoptotic function.