Maintenance of peripheral tolerance, while retaining the ability for initiation of immune responses is important for immune homeostasis. Induction of T cell tolerance and its in vitro counterpart anergy is an active process but the signaling events that predominate in anergy are different from those that predominate during productive responses. Although activation of Ras is blocked, activation of Rap1 is retained in anergic cells. To determine the mechanisms via which the active, GTP-bound Rap1 might affect the functional program of anergic cells we employed several approaches during the funding period of the last application: First, using the yeast two hybrid system we cloned RIAM, a novel Rapl-interacting molecule, which associates selectively with active, GTP-bound Rap1. RIAM provides a link between Rapl-GTP and the cytoskeleton by interacting with Mena, VASP and WASP and the actin regulator profilin. Second, by suppression subtractive hybridization we have determined that Rapl-GTP regulates gene transcription, resulting in a distinct gene expression pattern, which affects the function of T cells. Third, we observed that Rapl-GTP mediates cell spreading and adhesion to extracellular matrix via the Pl3 kinase pathway. Fourth, we have generated Rapl-GTP-Tg mice, which display impaired expression of T cell activation markers and defective T cell responses. These exciting results have initiated intensive in vitro and in vivo studies and have provided the experimental foundation for more than one research project. As a renewal of a R29, this grant will be subject to a budget cap that will support the project outlined below, but would not be sufficient for all of the scientific directions that we can now undertake. Therefore, studies on the function of RIAM, the Rapl-GTPIRIAM interactions, their effects on actin reorganization and the role of RIAM in T cell immunity and anergy will be continued in a separate project. For this application, we shall focus only on further exploration of the role of Rap-1 in T cell responses in vitro and in vivo. To achieve this objective two specific aims are proposed: First, to identify molecules, which are induced by or associate with Rap1 and study their functional activity in T cell responses in vitro and second, to determine the role of Rap1 in T cell responses in vivo by using mice transgenic for active or dominant negative Rap1.
