Activation of mitogen-activated protein kinase by the A(2A)-adenosine receptor via a rap1-dependent and via a p21(ras)-dependent pathway

The A(2A)-adenosine receptor, a prototypical G(s)-coupled receptor, activat es mitogen-activated protein (MAP) kinase in a manner independent of cAMP i n primary human endothelial cells. In order to delineate signaling pathways that link the receptor to the regulation of MAP kinase, the human A(2A) re ceptor was heterologously expressed in Chinese hamster ovary (CHO) and HEK2 93 cells. In both cell lines, A(2A) agonist-mediated cAMP accumulation was accompanied by activation of the small G protein rap1, However, rap1 mediat es A(2A) receptor-dependent activation of MAP kinase only in CHO cells, the signaling cascade being composed of G(s), adenylyl cyclase, rap1, and the p68 isoform of B-raf, This isoform was absent in HEK293 cells. Contrary to CHO cells, in HEK293 cells activation of MAP kinase by A(2A) agonists was n ot mimicked by 8-bromo-cAMP, was independent of G alpha(s), and was associa ted with activation of p21(ras), Accordingly, overexpression of the inactiv e S17N mutant of p21(ras) and of a dominant negative version of mSos (the e xchange factor of p21(ras)) blocked MAP kinase stimulation by the A(2A) rec eptor in HEK 293 but not in CHO cells. In spite of the close homology betwe en p21(ras) and rap1, the S17N\ mutant of rap1 was not dominant negative be cause (i) overexpression of rap1(S17N) failed to inhibit A(2A) receptor-dep endent MAP kinase activation, (ii) rap1(S17N) was recovered in the active f orm with a GST fusion protein comprising the rap1-binding domain of ralGDS after A(2A) receptor activation, and (iii) A(2A) agonists promoted the asso ciation of rap1(S17N) with the 68-kDa isoform of B-raf in CHO cells. We con clude that the A(2A) receptor has the capacity two activate MAP kinase via at least two signaling pathways, which depend on two distinct small G prote ins, namely p21(ras) and rap1, Our observations also show that the S17N ver sion of rap1 cannot be assumed a priori to act as a dominant negative inter fering mutant.