Agonists of the P2Y(AC)-receptor activate MAP kinase by a ras-independent pathway in rat C6 glioma.
We have previously shown that an ecto-NPPase modulates the ATP- and ADP-mediated P2Y(AC)-receptor activation in rat C6 glioma. In the present study, 2MeSADP and Ap(3)A induced no detectable PI turnover and were identified as specific agonists of the P2Y(AC)-receptor with EC(50) values of 250 +/- 37 pM and 1 +/- 0.5 microM, respectively. P2Y(AC)-receptor stimulation increased MAP kinase (ERK1/2) activation that returned to the basal level 4 h after stimulation and was correlated with a gradual desensitization of the P2Y(AC)-purinoceptor. The purinoceptor antagonists DIDS and RB2 blocked MAP kinase activation. An IP(3)-independent Ca(2+)-influx was observed after P2Y(AC)-receptor activation. Inhibition of this influx by Ca(2+)-chelation, did not affect MAP kinase activation. Pertussis toxin, toxin B, selective PKC-inhibitors and a specific MEK-inhibitor inhibited the 2MeSADP- and Ap(3)A-induced MAP kinase activation. In addition, transfection with dominant negative RhoA(Asn19) rendered C6 cells insensitive to P2Y(AC)-receptor-mediated MAP kinase activation whereas dominant negative ras was without effect. Immunoprecipitation experiments indicated a significant increase in the phosphorylation of raf-1 after P2Y(AC)-receptor activation. We may conclude that P2Y(AC)-purinoceptor agonists activate MAP kinase through a G(i)-RhoA-PKC-raf-MEK-dependent, but ras- and Ca(2+)-independent cascade.
Grobben
B
B
Department of Biochemistry, Cellular Biochemistry, Universiteit Antwerpen, Universitaire Instelling Antwerpen, Wilrijk, Belgium.
Claes
P
P
Van Kolen
K
K
Roymans
D
D
Fransen
P
P
Sys
S U
SU
Slegers
H
H
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D037342:Pertussis Toxin; D011493:Protein Kinase C / Q000502:physiology; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D018631:ras Proteins / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000502:physiology
D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins; D020742:rhoA GTP-Binding Protein; D000262:Adenylyl Cyclases