RIAM Activates Integrins by Linking Talin to Ras GTPase Membrane-targeting Sequences

Abstract

Rap1 small GTPases interact with Rap1-GTP-interacting adaptor molecule (RIAM), a member of the MRL (Mig-10/ RIAM/Lamellipodin) protein family, to promote talin-dependent integrin activation. Here, we show that MRL proteins function as scaffolds that connect the membrane targeting sequences in Ras GTPases to talin, thereby recruiting talin to the plasma membrane and activating integrins. The MRL proteins bound directly to talin via short, N-terminal sequences predicted to form amphipathic helices. RIAM-induced integrin activation required both its capacity to bind to Rap1 and to talin. Moreover, we constructed a minimized 50-residue Rap-RIAM module containing the talin binding site of RIAM joined to the membrane-targeting sequence of Rap1A. This minimized Rap-RIAM module was sufficient to target talin to the plasma membrane and to mediate integrin activation, even in the absence of Rap1 activity. We identified a short talin binding sequence in Lamellipodin (Lpd), another MRL protein; talin binding Lpd sequence joined to a Rap1 membrane-targeting sequence is sufficient to recruit talin and activate integrins. These data establish the mechanism whereby MRL proteins interact with both talin and Ras GTPases to activate integrins.Increased affinity ("activation") of cellular integrins is central to physiological events such as cell migration, assembly of the extracellular matrix, the immune response, and hemostasis (1). Each integrin comprises a type I transmembrane ␣ and ␤ subunit, each of which has a large extracellular domain, a single transmembrane domain, and a cytoplasmic domain (tail). Talin binds to most integrin ␤ cytoplasmic domains and the binding of talin to the integrin ␤ tail initiates integrin activation (2-4). A small, PTB-like domain of talin mediates activation via a twosite interaction with integrin ␤ tails (5), and this PTB domain is functionally masked in the intact talin molecule (6). A central question in integrin biology is how the talin-integrin interaction is regulated to control integrin activation; recent work has implicated Ras GTPases as critical signaling modules in this process (7).Ras proteins are small monomeric GTPases that cycle between the GTP-bound active form and the GDP-bound inactive form. Guanine nucleotide exchange factors (GEFs) promote Ras activity by exchanging bound GDP for GTP, whereas GTPase-activating proteins (GAPs) 3 enhance the hydrolysis of Ras-bound GTP to GDP (for review, see Ref. 8). The Ras subfamily members Rap1A and Rap1B stimulate integrin activation (9, 10). For example, expression of constitutively active Rap1 activates integrin ␣M␤2 in macrophage, and inhibition of Rap1 abrogated integrin activation induced by inflammatory agonists (11-13). Murine T-cells expressing constitutively active Rap1 manifest enhanced integrin dependent cell adhesion (14). In platelets, Rap1 is rapidly activated by platelet agonists (15, 16). A knock-out of Rap1B (17) Recently we used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway...

Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

“…In the literature, the Rap effector Rap1-GTP-interacting adapter molecule (RIAM) has been established as a direct link between Rap and cell-extracellular matrix (ECM) adhesion, as overexpression of RIAM induces integrin activation and cell adhesion, and the depletion of RIAM abrogates Rap1 induced adhesion. 65,66 As we do not identify any 007-regulated phosphoproteins that more directly link Rap with the structural components involved in cell-ECM adhesion, such as focal adhesions and integrins, this may well indicate that phosphorylation is not involved in the initial phase of Rap-mediated cell-substrate adhesion. In addition, from our pathway and GO term analyses, we can observe that the biological processes that are affected upon Rap activation include cell-cell adhesion, cell-substratum adhesion, and (Rho GTPase-regulated) actincytoskeleton rearrangements, which is in line with the results from the STRING network analysis.…”

The tale of two talins – two isoforms to fine‐tune integrin signalling

“…In fibroblasts, both talins localise to FAs, and talin1 is recruited directly to the leading edge, via proteins like the Rap1 effector RIAM (Rap1‐interacting adapter molecule) 29, 30 and FAK (focal adhesion kinase) 31. In contrast, less is known about talin2 recruitment.…”

“…This mode of binding to talin was initially identified from work on the tumour suppressor protein deleted in liver cancer 1 (DLC1) 94, 95. This led on to the identification of the talin‐binding sequence in RIAM 24, 29 as an LD‐motif, and the identification of paxillin as a novel talin ligand 94. More recently, the KANK (kidney ankyrin repeat‐containing) proteins have been identified as LD‐motif‐containing ligands 16, 17, binding to a conserved face on the R7 5‐helix bundle.…”

Subcellular Localization of Talin Is Regulated by Inter-domain Interactions

“…Talin-As noted above, THD contains multiple lipid-binding sites that could support membrane interactions (21,22,26,31), and previous studies raised the possibility that such sites within FL talin might be masked by the talin rod domain (17)(18)(19).…”

“…Furthermore, recent studies using model cellular systems have established that talin is cytosolic until it interacts with an activated Rap1-RIAM complex that recruits FL talin to the plasma membrane (17)(18)(19). These data suggest that talin might be autoinhibited to regulate its localization and thus interactions with integrins and resulting biological functions.…”