The structural basis of Arfaptin-mediated cross-talk between Rac and Arf signalling pathways

Small G proteins are GTP-dependent molecular switches that regulate numerou s cellular functions(1). They can be classified into homologous subfamilies that are broadly associated with specific biological processes. Cross-talk between small G-protein families has an important role in signalling, but the mechanism by which it occurs is poorly understood(2). The coordinated a ction of Arf and Rho family GTPases is required to regulate many cellular p rocesses including lipid signalling(3), cell motility(4) and Golgi function (5). Arfaptin is a ubiquitously expressed protein implicated in mediating c ross-talk between Rac (a member of the Rho family) and Arf small GTPases. H ere we show that Arfaptin binds specifically to GTP-bound Arf1 and Arf6, bu t binds to Rac.GTP and Rac.GDP with similar affinities. The X-ray structure of Arfaptin reveals an elongated, crescent-shaped dimer of three-helix coi led-coils. Structures of Arfaptin with Rac bound to either GDP or the slowl y hydrolysable analogue GMPPNP show that the switch regions adopt similar c onformations in both complexes. Our data highlight fundamental differences between the molecular mechanisms of Rho and Ras family signalling, and sugg est a model of Arfaptin-mediated synergy between the Arf and Rho family sig nalling pathways.