ABSTRACT
The small GTPase Rap1 controls the actin cytoskeleton by regulating Rho GTPase signaling. We recently established that the Rap1 effectors Radil and Rasip1, together with the Rho GTPase activating protein ArhGAP29, mediate Rap1-induced inhibition of Rho signaling in the processes of epithelial cell spreading and endothelial barrier function. Here, we show that Rap1 induces the independent translocations of Rasip1 and a Radil-ArhGAP29 complex to the plasma membrane. This results in the formation of a multimeric protein complex required for Rap1-induced inhibition of Rho signaling and increased endothelial barrier function. Together with the previously reported spatiotemporal control of the Rap guanine nucleotide exchange factor Epac1, these findings elucidate a signaling pathway for spatiotemporal control of Rho signaling that operates by successive protein translocations to and complex formation at the plasma membrane.
Subject(s)
GTPase-Activating Proteins/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Signal Transduction , rap1 GTP-Binding Proteins/metabolism , rho GTP-Binding Proteins/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cells, Cultured , GTPase-Activating Proteins/genetics , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , HEK293 Cells , Humans , Intercellular Junctions/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Confocal , Protein Binding , Protein Transport , RNA Interference , rap1 GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/geneticsABSTRACT
In the present study a unique antibody (NKI-L16) reacting with the alpha-chain of the human leukocyte function-associated Ag-1 (LFA-1) is described, which stimulates homotypic cell-cell interactions in a manner very similar to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), in contrast to other anti-LFA-1 mAb which inhibit cell aggregation. The induction of aggregate formation of EBV-transformed B cells (JY) and CTL clones by TPA or NKI-L16 is not accompanied by an increase in the expression of LFA-1. Nevertheless, this cluster formation is LFA-1 dependent, inasmuch as anti-LFA-1 antibodies, other than NKI-L16, completely abrogate aggregation. Simultaneous addition of NKI-L16 and TPA did not result in a further increase of the speed of cluster formation, suggesting that a similar pathway is activated. Immunoprecipitation and enzyme digestion studies revealed that NKI-L16 recognizes a unique epitope on the alpha-chain of LFA-1, most likely situated close to the transmembrane segment of the molecule. It is hypothesized that NKI-L16 or TPA can cause the LFA-1 molecule to convert from an inactive to an active configuration, thereby permitting binding of LFA-1 to its natural ligand.
