Binding of the PH and polybasic C-terminal domains of ARNO to phosphoinositides and to acidic lipids

The activity on ARF of the guanine nucleotide exchange factor ARNO depends on its membrane recruitment, induced by binding of its PH domain to phospho inositides. A polycationic C-terminal extension to the PN domain might also contribute to its specific binding to phosphatidylinositol 4,5-bisphosphat e [(4,5)PIP2] and to phosphatidylinositol 3,4,5-trisphosphate [(3,4,5)PIP3] , and to ionic binding to other acidic lipids. We have analyzed in vitro th e relative contributions to phospholipid binding of the PH domain and C-ter minal extension by cosedimentation of "PH+C domain" and "nominal PH domain" protein constructs including or not including the polycationic C-terminus, with sucrose-loaded unilamellar vesicles made of equal proportions of the neutral lipids phosphatidylcholine and phosphatidylethanolamine, and supple mented or not with 30% acidic phosphatidylserine (PS) and 2% of various pho sphoinositides. Binding was measured as a function of the vesicle concentra tion and of the medium ionic strength. Both proteins bound with higher affi nity to (3,4,5)PIP3 than to (4,5)PIP2, the selectivity for (3,4,5)PIP3 bein g highest for the nominal PH domain. We observed also a clear selectivity o f (3,4,5)PIP3 over (4,5)PIP2 for stimulating the activity of ARNO on ARF wi th vesicles containing 10% PS and 1% PIP:! or PIP3. Our data suggest that t he PH domain provides the specific phosphoinositide binding site and some u nspecific ionic interaction with acidic PS, whereas the polybasic C domain contributes to binding mainly by unspecific ionic interactions vith PS. Pho sphorylation by protein kinase C of a serine in the C domain reduces the io nic affinity of the PH+C domain for PS, but does not affect the phosphoinos itide specificity.