A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3 , whose function is required for both chemotaxis and the synthesis and rela y of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unab le to aggregate and lack receptor activation of adenylyl cyclase but are ab le, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. Ln addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotax is is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyc lase, which is required for chemotaxis, is reduced similar to 60% in rip3 n ull cells. This reduced activation of guanylyl cyclase may account, in part , for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound st age. Unlike the response seen in wild-type strains, the rip3 null cell aggr egates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenot ypes of the rip3 null cell, including the inability to activate adenylyl cy clase in response to cAMP and defects in chemotaxis, are very similar to th ose of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a de fect in cAMP-mediated activation of guanylyl cyclase similar to that of rip 3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a fu rther reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras p rotein RasG, which itself is important for cell movement, we propose that R IP3 and AleA are components of a Ras regulated pathway involved in integrat ing chemotaxis and signal relay pathways that are essential for aggregation .