ADP-ribosylation factor 6 as a target of guanine nucleotide exchange factor GRP1

The GRP1 protein contains a Sec7 homology domain that catalyzes guanine nuc leotide exchange on ADP-ribosylation factors (ARF) 1 and 5 as well as a ple ckstrin homology domain that binds phosphatidylinositol(3,4,5)P-3, an inter mediate in cell signaling by insulin and other extracellular stimuli (Klarl und, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that both endogen ous GRP1 and ARF6 rapidly co-localize in plasma membrane ruffles in Chinese hamster ovary (CHO-T) cells expressing human insulin receptors and COS-1 c ells in response to insulin and epidermal growth factor, respectively. The pleckstrin homology domain of GRP1 appears to be sufficient for regulated m embrane localization. Using a novel method to estimate GTP loading of expre ssed HA epitope-tagged ARF proteins in intact cells, levels of biologically active, GTP-bound ARF6 as well as GTP-bound ARF1 were elevated when these ARF proteins were co-expressed with GRP1 or the related protein cytohesin-1 . GTP loading of ARF6 in both control cells and in response to GRP1 or cyto hesin-1 was insensitive to brefeldin A, consistent with previous data on en dogenous ARF6 exchange activity. The ability of GRP1 to catalyze GTP/GDP ex change on ARF6 was confirmed using recombinant proteins in a cell-free syst em. Taken together, these results suggest that phosphatidylinositol(3,4,5)P -3 may be generated in cell membrane ruffles where receptor tyrosine kinase s are concentrated in response to growth factors, causing recruitment of en dogenous GRP1. Further, co-localization of GRP1 with ARF6, combined with it s demonstrated ability to activate ARF6, suggests a physiological role for GRP1 in regulating ARF6 functions.