30] Isolation and properties of GRP1, an ADP-ribosylation factor (ARF)-guanine nucleotide exchange protein regulated by phosphatidylinositol 3,4,5-triphosphate

Abstract

This chapter discusses the isolation and properties of general receptor for 3-phosphoinositides (GRP1), an adenosine diphosphate (ADP)-ribosylation factor (ARF)-guanine nucleotide exchange protein regulated by phosphatidylinositol 3,4,5-trisphosphate. GRP1 was isolated based on the high affinity binding to PtdIns(3,4,5)P 3 of its PH domain, using a screen of a cDNA expression library. The Sec7 homology domain of GRP1 appears to be capable of catalyzing GTP-GDP exchange on all known ARF proteins. Direct demonstration 2,5 of such activity in cell-free systems has been reported for ARF1, ARF5, and ARF6 proteins, which represent each of the three classes of known ARF proteins. This suggests that the related ARF2, ARF3, and ARF4 are also targets of GRP1. However, initially it was observed that ARF6 was apparently not a substrate for GRP15 or ARNO. Subsequently, using a different assay method, two groups showed that ARF6 is indeed a good substrate for these proteins as well. Both assays used are excellent, however, when used with ARF1, and therefore both are provided in this chapter.