Purification and properties of Rab3 GEP (DENN/MADD)

Rab3A, a member of the Rab3 small GTP-binding protein (G protein) family, regulates Ca(2+)-dependent exocytosis of neurotransmitter. Rab3A cycles between the GDP-bound inactive and GTP-bound active forms, and the former is converted to the latter by the action of a GDP/GTP exchange protein (GEP). We have previously purified a GEP from rat brain with lipid-modified Rab3A as a substrate. Purified Rab3 GEP is active on all the Rab3 subfamily members including Rab3A, -3B, -3C, and -3D. Purified Rab3 GEP is active on the lipid-modified form, but not on the lipid-unmodified form. Purified Rab3 GEP is inactive on Rab3A complexed with Rab GDI. The recombinant protein is prepared from the Rab3 GEP-expressed Spodoptera frugiperda cells (Sf9 cells). The properties of recombinant Rab3 GEP, including the requirement for lipid modifications of Rab3A, the substrate specificity, and the sensitivity to Rab GDI, are similar to those of purified Rab3 GEP. Overexpression of Rab3 GEP inhibits Ca(2+)-dependent exocytosis from PC12 cells. On the other hand, Rab3 GEP is identical to a protein named DENN/MADD: differentially expressed in normal versus neoplastic (DENN)/mitogen-activated protein kinase-activating death domain (MADD). Here, we describe the purification method for recombinant Rab3 GEP from Sf9 cells and the functional properties of Rab3 GEP in Ca(2+)-dependent exocytosis by use of the human growth hormone coexpression assay system of PC12 cells.