The N-terminus of Af6 was identified as the Ras-binding site. Additionally to the Rasbinding Af6 also binds Rap. ZO-1, a protein involved in the formation of tight junctions, also binds to Af6 close to the amino terminus thereby competing with Ras binding. This data suggests a participation of Af6 in the regulation of cell-cell contacts via a Rasmodulated interaction with ZO-1. In contrast to the well characterized effectors Raf, RalGEF, and PI(3)kinase, Af6 is a protein with no enzymatic function. It seems to serve as a scaffolding component for protein complexes. This would represent a new type of Ras effector because Ras binding in this case does not lead to the activation of the enzymatic activity of the effector.

The GST-Tag facilitates the protein‘s application in typical GST pull down assays.