Adenosine nucleotides acting at the human P2Y1 receptor stimulate mitogen-activated protein kinases and induce apoptosis.
For the widely distributed P2Y receptors for nucleotides, the transductional and functional responses downstream of their coupling to G proteins are poorly characterized. Here we describe apoptotic induction and the associated 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) (EC(50) approximately 5 nm) as well as several, but not all isoforms detected, of the stress-activated protein kinase (SAPK) family. Phospho-isoforms of p38 were unaffected. The induced kinase activity was blocked by the P2Y(1) receptor-selective antagonist, adenosine-2'-phosphate-5'-phosphate, but unaffected by pertussis toxin. In addition, the endogenous ligand ADP, and significantly also 2-MeSATP, induced concentration-dependent phosphorylation changes in the same MAP kinase family members. The sustained activation of ERK1/2 was associated with Elk-1 phosphorylation that was abolished by the MEK1 inhibitor, PD 98059. However, the concomitant transient activation of the SAPKs was not sufficient to induce c-Jun or ATF-2 phosphorylation. The transient phase of the ERK activity was partially inhibited either by the phosphatidylinositol 3-kinase inhibitor, LY 294002, or the PKC inhibitor, Gö 6976. In addition, the Src inhibitor, PP1, or expression of dominant negative Ras also attenuated the transient phase of ERK phosphorylation. In contrast, inhibition of Ras or Src had no effect on the sustained ERK activity, which was critically dependent on phosphatidylinositol 3-kinase. 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 human P2Y(1) receptor can transiently activate the Ras-ERK cascade via the cooperative effects of phosphatidylinositol 3-kinase, Src and PKC. The sustained ERK stimulation, via a Ras-insensitive pathway, culminates in Elk-1 activation without inducing a proliferation effect. The transient SAPK activity did not evoke transcription factor phosphorylation but was required for the P2Y(1) receptor-mediated apoptotic function.
Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, Cambridge CB2 1QJ, United Kingdom.
L A
LA
Sellers
J
J
Simon
T S
TS
Lundahl
D J
DJ
Cousens
P P
PP
Humphrey
E A
EA
Barnard
D000227:Adenine Nucleotides / Q000494:pharmacology*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001254:Astrocytoma; D002217:Carbachol / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D037342:Pertussis Toxin; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000502:physiology*; D058472:Receptors, Purinergic P2Y1; D011994:Recombinant Proteins / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology; D014157:Transcription Factors; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D050807:ets-Domain Protein Elk-1; D048051:p38 Mitogen-Activated Protein Kinases
D000227:Adenine Nucleotides; D017304:Annexin A5; D002227:Carbazoles; D002867:Chromones; D004268:DNA-Binding Proteins; C494112:ELK1 protein, human; D004791:Enzyme Inhibitors; D007211:Indoles; D009025:Morpholines; C546382:P2RY1 protein, human; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D011994:Recombinant Proteins; D013873:Thionucleotides; D014157:Transcription Factors; D010566:Virulence Factors, Bordetella; D050807:ets-Domain Protein Elk-1; C081021:Go 6976; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C002480:methylthio-ADP; D000244:Adenosine Diphosphate; D002217:Carbachol; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins