Heterotrimeric G protein-independent signaling of a G protein-coupled receptor

The A(2A) adenosine receptor is a prototypical G(s)-coupled receptor, but it also signals, e. g. to mitogen-activated protein ( MAP) kinase, via a pathway that is independent of heterotrimeric G proteins. Truncation of the carboxyl terminus affects the strength of the signal through these alternative pathways. In a yeast two-hybrid interaction hunt, we screened a human brain library for proteins that bound to the juxtamembrane portion of the carboxyl terminus of the A(2A) receptor. This approach identified ARNO/cytohesin-2, a nucleotide exchange factor for the small ( monomeric) G proteins of the Arf ( ADP-ribosylation factor) family, as a potential interaction partner. We confirmed a direct interaction by mutual pull down ( of fusion proteins expressed in bacteria) and by immunoprecipitation of the proteins expressed in mammalian cells. To circumvent the long term toxicity associated with overexpression of ARNO/cytohesin-2, we created stable cell lines that stably expressed the A(2A) receptor and where ARNO/cytohesin-2 or the dominant negative version E156K-ARNO/cytohesin-2 was inducible by mifepristone. Cyclic AMP accumulation induced by an A(2A)-specific agonist was neither altered by ARNO/ cytohesin-2 nor by the dominant negative version. This was also true for agonist-induced desensitization. In contrast, expression of dominant negative E156K-ARNO/cytohesin-2 and of dominant negative T27N-Arf6 abrogated the sustained phase of MAP kinase stimulation induced by the A(2A) receptor. We therefore conclude that ARNO/ cytohesin-2 is required to support the alternative, heterotrimeric G protein-independent, signaling pathway of A(2A) receptor, which is stimulation of MAP kinase.