Activation of MAP kinase by the A_2A-Adenosine receptor

摘要

The A2A-adenosine receptor, a prototypical Gs-coupled receptor, activates MAP kinase in a manner independent of cAMP in primary human endothelial cells. In order to delineate signalling pathways that link the receptor to the regulation of MAP kinase, the human A_2A-receptor was heterologously expressed in CHO and HEK293 cells. In both cell lines, A_2A-agonist mediated cAMP accumulation was accompanied by activation of the small G protein rap1; however, rap1 mediates A_2A-receptor-dependent MAP kinase activation only in CHO cells (the signalling cascade being composed of G_s, adenylyl cyclase, protein kinase A, rapl and B-raf). The S17N-mutant of rapl was not dominant negative; (i) following transient expression, rapl(S17N) failed to inhibit receptor-dependent MAP kinase activation, (ii) After receptor-activation, rapl(S17N) was recovered in the active form with a GST(glutathionS-transferase)-fusion protein comprising the rapl-binding domain of ralGDS. (iii) In CHO cells, A_2A-agonists promoted the association of rapl(S17N) with B-raf. In HEK293 cells, activation of MAP kinase by A_2A-agonists required p21~(ras), because it was blocked after expression of the dominant negative version ras(S17N); in addition, an increase active p21~(ras) (detected by pull-downs with a GST-fusion protein encompassing the ras-binding domain of raf-1) was seen after A2A-receptor stimulation. We conclude that the A_2A-receptor has the capacity to activate MAP kinase via at least two signalling pathways.