The N-terminal coiled-coil domain of the cytohesin/ARNO family of guanine nucleotide exchange factors interacts with G alpha q

摘要

Cytohesins are guanine-nucleotide exchange factors (GEF) for the Arf family of GTPases. One member of the Arf family, ARF6, plays an active role in the intracellular trafficking of G protein-coupled receptors. We have previously reported that G alpha q signaling leads to the activation of ARF6, possibly through a direct interaction with cytohesin-2/ARNO. Here, we report that G alpha q can directly interact with cytohesin-1, another Arf-GEF of the ARNO/cytohesin family. Cytohesin-1 preferentially associated with a constitutively active mutant of G alpha q (G alpha q-Q209L) compared to wild-type G alpha q in HEK293 cells. Stimulation of TP beta, a G alpha q-coupled receptor, to activate G alpha q resulted in the promotion of a protein complex between G alpha q and cytohesin-1. Confocal immunofluorescence microscopy revealed that wild-type G alpha q and cytohesin-1 co-localized in intracellular compartments and at or near the plasma membrane. In contrast, expression of G alpha q-Q209L induced a drastic increase in the localization of cytohesin-1 at the plasma membrane. Expression of a dominant-negative mutant of cytohesin-1 reduced by 40% the agonist-induced internalization of TP beta, a process that we previously demonstrated to be dependent on G alpha q-mediated signaling and Arf6 activation. Using deletion mutants, we show that cytohesin-1 interacts with G alpha q through its N-terminal coiled-coil domain. Cytohesin-1 and cytohesin-2/ARNO mutants lacking the coiled-coil domain were unable to relay G alpha q-mediated activation of Arf6. This is the first report of an interaction between the coiled-coil domain of the cytohesin/ARNO family of Arf-GEFs and a member of the heterotrimeric G proteins.