REGULATION OF GRP1-CATALYZED ADP-RIBOSYLATION FACTOR GUANINE-NUCLEOTIDE EXCHANGE BY PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE

Cellular levels of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3, 4,5)P-3) are rapidly elevated in response to activation of growth fact or receptor tyrosine kinases. This polyphosphoinositide binds the plec kstrin homology (PH) domain of GRP1, a protein that also contains 200 residues with high sequence similarity to a segment of the yeast Sec7 protein that functions as an ADP ribosylation exchange factor (ARF) (K larlund, J., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A. and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that di octanoyl PtdIns(3,4,5)P, binds the PH domain of GRP1 with a K-d = 0.5 mu M, an affinity 2 orders of magnitude greater than dioctanoyl-PtdIns (4,5)P-2. Further, the Sec7 domain of GRP1 is found to catalyze guanin e nucleotide exchange of ARF1 and -5 but not ARF6. Importantly, PtdIns (3,4,5)P-3, but not PtdIns(4,5)P-2, markedly enhances the ARF exchange activity of GRP1 in a reaction mixture containing dimyristoylphosphat idylcholine micelles, cholamidopropyl)dimethylammonio]-1-propanesulfon ic acid, and a low concentration of sodium cholate. PtdIns(3,4,5)P-3-m ediated ARF nucleotide exchange through GRP1 is selectively blocked by 100 mu M inositol 1,3,4,5-tetrakisphosphate, which also binds the PH domain of GRP1. Taken together, these data are consistent with the hyp othesis that selective recruitment of GRP1 to PtdIns(3,4,5)P-3 in memb ranes activates ARF1 and -5, known regulators of intracellular membran e trafficking.