Signal transduction of transfected and native P2Y receptors

• Main Author: Roberts, Jonathan A.
• Published: University of Leicester 1998
• Subjects: 571.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.696516

This thesis looks at the turkey P2Y1, human P2Y2, human P2Y4 and rat P2Y6 receptors transfected into the human astrocytoma null cell line 1321N1 to investigate signalling pathways linking G protein receptor activation to tyrosine phosphorylation, MAPK and mitogenesis. This was compared with results for the native primary cell preparation of rat brain microvascular endothelial cells. Previous work by others and us has established that all 1321N1 transfected P2Y receptors are strongly linked to an increase in PLC activation. Neither the turkey P2Y1 or human P2Y2 receptors were coupled to an increase in overall tyrosine phosphorylation assessed by PY20 antibody western blot. Pervanadate alone gave large increases in tyrosine phosphorylation, but no further increase in tyrosine phosphorylation was observed with co-addition of 2MeSATP to the turkey P2Y1 transfectants. Co-addition of UTP and a sub-maximal concentration of prevanadate on the human P2Y2 receptor gave a reduction in tyrosine phosphorylation compared to pervanadate alone. This indicated possible activation of tyrosine phosphatase activity by the human P2Y1 and human P2Y2 receptors were both shown to activate p42/p44 MAPK assessed by phospho-MAPK antibody western blotting and a nonapeptide kinase assay. Both turkey P2Y1 and human P2Y2 receptor activation of MAPK was inhibited by the MEK inhibitor PD 98059. Human P2Y4 and rat P2Y6 receptors showed no activation of MAPK. Both turkey P2Y1 and human P2Y2 MAPK activation was PKC dependant; inhibited by Ro 31-8220 and Go 6850, but not Go 6976 a calcium sensitive PKC isoform inhibitor. PKC isoforms ( or ) may be involved in this signalling pathway. Some experiments investigating Pyk2 and Shc involvement in P2Y signalling are presented.