Enhancing activity and phospholipase A(2) activity: two independent activities present in the enhancing factor molecule

Enhancing factor (EF), a molecule that increases the binding of epidermal g rowth factor (EGF) to A431 cells, was first isolated in our laboratory from mouse intestines, and subsequently shown to be a secretory form of phospho lipase A(2)(PLA(2)) [Mulherkar, Rao, Wagle, Patki and Deo (1993) Biochem. B iophys. Res. Commun. 195, 1254-1263]. We had proposed earlier that EF incre ases the binding of EGF by first binding to its own cell-surface receptor [ identified as a 100 kDa molecule; Mulherkar and Deo (1986) J. Cell. Physiol . 127, 183-188], and then by creating a binding site for EGF. However, due to its PLA(2) activity, there was a possibility that EF, by its phospholipa se activity could be unmasking cryptic EGF receptors on the cell surface, t hereby increasing the number of binding sites for EGF. To test whether enha ncing activity and phospholipase activity are independent of each other, a series of mutations were created using the full-length EF cDNA as a templat e, expressed in 293 cells and the mutant recombinant proteins checked for E F as well as PLA(2) activities. Our studies have shown that one of the muta nt EF proteins, lacking PLA(2) activity, retains EF activity. This demonstr ates unambiguously that EF and PLA(2) activities are two independent activi ties in the same molecule. Mutation in the Ca2+-binding loop resulted in lo ss of EF activity, thereby demonstrating that EF activity is Ca2+-dependent . The N-terminal region of the EF molecule appears to be crucial for the en hancing activity.