RIAM, a New Rap1 Effector, Functions Downstream of Rap1 and Regulates Rap1 Localization at the Plasma Membrane and Rap1-Induced Adhesion.
Authors: Lafuente Esther M. , Carman Christopher V. , Krause Matthias , Springer Timothy A. , Gertler Frank B. , Boussiotis Vassiliki A. 
Published: October 2019

Abstract
The small GTPase Rap1, originally identified by its ability to reverse Ras-mediated transformation, regulates cytoskeleton organization, adherens junction positioning and cell adhesion. In T cells, Rap1 is activated upon TCR ligation and recruited to the plasma membrane where it regulates activation of β1 (VLA4, VLA5) and β2 (LFA-1) integrins. However, it is poorly understood how active Rap1 is localized at the plasma membrane and how it mediates integrin activation. Recently, we identified RIAM, a novel adaptor molecule that interacts specifically with active GTP-bound Rap1 and with regulators of the actin cytoskeleton Evl, VASP and Profilin. Because Rap1-GTP induces integrin mediated adhesion, we examined whether RIAM might have a similar effect. Stable Jurkat T cell lines transfected with either RIAM, the GTP-bound mutant Rap1E63 or empty vector, were added in plates coated with fibronectin or ICAM-1. Cells transfected with RIAM had increased adhesion, which was comparable to that induced by Rap1E63. RIAM- and Rap1E63-induced adhesion on fibronectin was blocked by an antibody against the β1 chain of VLA-4, whereas adhesion on ICAM-1 was blocked by a blocking LFA-1 mAb. To determine whether RIAM induced active conformational changes in beta1 integrins, we analyzed activation epitope exposure by incubation with HUTS4 antibody that specifically binds to β1 integrins in their active conformation. RIAM transfected cells showed increased binding to HUTS4. RIAM also induced active conformation of LFA-1 as determined by using KIM127 antibody that specifically binds to β2 integrins in their active conformation. The fact that RIAM interacts selectively with Rap1-GTP and induces integrin activation suggests that RIAM functions as a Rap1-downstream effector. To test this hypothesis we generated stable Jurkat T cell lines expressing Rap1E63, and either specific RIAM shRNAs to knockdown expression of endogenous RIAM (Rap1E63-RIAM-KD) or shRNA control (Rap1E63-control-KD). Compared to Rap1E63, Rap1E63-RIAM-KD cells lost their enhanced adhesion properties and displayed reduced adhesion to fibronectin and ICAM-1 to levels similar to parental control Jurkat cells. In contrast, Rap1E63-control-KD retained increased adhesion to fibronectin and ICAM-1, to levels similar to Rap1E63 cells. Rap1 is recruited to the plasma membrane upon activation and this localization is required for Rap1-GTP to mediate cell adhesion. Therefore, we examined whether, similarly to adhesion, distribution of Rap1-GTP was affected by depletion of RIAM. Rap1E63-RIAM-KD and Rap1E63-control-KD Jurkat cells were seeded on anti-CD3 coated slides, fixed, probed with GST-RalGDS-RBD that interacts specifically with Rap1-GTP, and analyzed by confocal microscopy. In Rap1E63-control-KD cells Rap1-GTP was detected at the perinuclear region and at the plasma membrane, where it co-localized with polymerized (F)-actin. Strikingly, in Rap1E63-RIAM-KD cells Rap1-GTP was displaced from the plasma membrane and was detected only in the perinuclear region. In these cells no co-localization of Rap1-GTP with F-actin was detected. Thus, RIAM is a critical downstream effector of Rap1, which regulates Rap1-GTP localization at the plasma membrane and mediates active conformation of integrins and Rap1-induced adhesion.