Effectors of small GTPases are defined as signaling molecules that bind to the GTPase only when it is in the GTP-bound configuration and, in so doing, affect a change that conveys signaling information. The signaling information may be allosteric regulation of the effector, if it is an enzyme, or recruitment of the effector to the relevant subcellular compartment. In the case of RIAM, it appears that relocalization from the cytosol to the PM is the principle function of its RA domain. This is also true for the best studied Ras effector, Raf-1, which is recruited to the PM via its RA domain/RBD. In the case of Raf-1, it is clear that the RBD itself is necessary and sufficient to drive Raf-1 to membranes decorated with activated GTP-bound Ras (Chiu et al., 2002
). In contrast, our data show that the RA domain of RIAM is not sufficient but requires an additional contribution from a PH domain that is structurally integrated with the RA domain.
Our inability to detect PM translocation of full-length GFP-tagged RIAM to the PM of Jurkat cells by live cell imaging cannot be interpreted as indicating that endogenous RIAM does not associate with the PM. This is a common result with GFP-tagged, overexpressed, multidomain signaling proteins because when the pool that translocates is small relative to the vast amount that remains in the cytosol, it is difficult to score for PM enhancement by fluorescence. This is the case for PM recruitment of the Ras effector Raf-1 (Bondeva et al., 2002
; Chiu et al., 2002
). Our analysis of endogenous RIAM by subcellular fractionation () confirms that only a small portion of the protein stably associates with membranes in stimulated cells.
Both Ras and Rap proteins are present on multiple subcellular compartments (Choy et al., 1999
; Bivona et al., 2004
). The RA domains of Raf-1 and RalGDS bind Ras and Rap, respectively, with relatively high affinity and have been shown to colocalize with the activated form of the GTPase on all membrane compartments on which they are expressed (Chiu et al., 2002
; Bivona et al., 2004
). This observation has led to the recognition that Ras proteins signal from multiple subcellular compartments and that the signal output differs from each localization, providing an additional level of signaling complexity (Chiu et al., 2002
). Our data show that, in contrast, signaling through RIAM is restricted to only one of the compartments upon which GTP-bound Rap1 accumulates. The affinity of the RIAM PH domain for PI(4,5)P2
, a PIP which is enriched only in the inner leaflet of the PM, explains the spatial restriction of RIAM membrane association. Whereas Raf-1 regulates MEK/Erk kinases, which have roles both at the PM and in the cytoplasm (Torii et al., 2004
), RIAM regulates LFA-1, which functions only at the PM. Thus, the spatial restriction afforded by combining a relatively low affinity RA domain with a PI(4,5)P2
-directed PH domain would provide efficiency by bringing RIAM only to the subcellular localization where it is needed.
RA domains/RBDs possess a ubiquitin fold and typically bind to Ras proteins through an inter-protein β-sheet stabilized by electrostatic interactions (Emerson et al., 1995
; Nassar et al., 1995
). Importantly, the binding affinities for GTP-bound GTPases vary widely, with Kd
values that range from 80 nM for Raf-1/Ras to 4 µM for RIN1/Rap1 (Wohlgemuth et al., 2005
). Our in vitro pulldown experiments () show that RIAM RA-PH binds to GTP-loaded Rap1 or H-Ras with relatively low and approximately equal affinities. Previous structural and mutagenesis studies pinpointed residue 31—Glu31 in Ras and Lys31 in Rap1—as a key specificity determinant in the binding of the RA domains of Raf-1 and RalGDS (Nassar et al., 1996
). Lys84 (α1) in the Raf-1 RA domain interacts electrostatically with Glu31 of Ras, and Asp56 (α1-β3 loop) in RalGDS interacts electrostatically with Lys31 of Rap1. In RIAM and the other RA-PH proteins, the residues corresponding to Lys84 (Raf-1) and Asp 56 (RalGDS) are not charged (and not conserved), providing a rationale as to why the RA-PH proteins are less discriminating in binding to Ras versus Rap1.
The crystal structure of the RIAM RA and PH domains shows that these two domains, separated in sequence by ~50 residues, nevertheless physically associate through a conserved interface to form a single structural unit. This feature was also observed in the crystal structure of the RA-PH domains of Grb10 (Depetris et al., 2009
), suggesting that it will be a general feature of the MRL and Grb7/10/14 protein families.
The integrated structural nature of the RA-PH domains (and the hydrophobic interface) suggests that they are unlikely to fold properly when expressed individually. This conclusion is supported by a recent study showing that the RA domain of RIAM is unstable without the PH domain (Takala and Ylänne, 2012
). These observations force a reinterpretation of results in published studies using the RA or PH domains expressed alone (Ménasché et al., 2007
) and suggested that we could not readily express the RA and PH domains separately to parse their individual contributions to membrane binding. To circumvent this problem, we expressed tandem RIAM RA-PH domains in which combinations of the binding motifs could be rendered nonfunctional by point mutations. These experiments revealed important characteristics of the two membrane association domains. First, whereas the tandem RA domains separated by a flexible linker could not anchor GFP at the PM, the tandem PH domains could. This suggests that the added affinity and avidity of dual PH domains is sufficient for binding to endogenous levels of PI(4,5)P2
, whereas even tandem RA domains fall below the affinity threshold for membranes that display activated Rap1. Besides simple differences in affinity, the abundance and mobility of GTP-bound Rap1 and PI(4,5)P2
in the plane of the membrane are likely to be markedly different, which may contribute to the different relative contributions of the two binding domains.
Second, and more important, experiments with duplicate RA-PH domains demonstrated that the RIAM RA-PH integrated structural unit is more efficient at colocalizing with activated Rap1 than are separated RA and PH domains. Having dual membrane targeting motifs constitutes a proximity detector that affords a higher level of regulation than can be achieved with a single binding motif (Lemmon, 2008
). It seems plausible that the evolutionary advantage of the structurally integrated RA-PH domains is that the interaction of the PH domain with membrane enriched in PI(4,5,)P2
spatially positions the RA domain for facile interaction with PM-tethered Rap1. In a beads-on-a-string model, with the PH domain bound to the PM, the independent RA domain would adopt multiple positions and orientations, some of which would not be compatible with Rap1 binding.
Our data clearly show that the isolated RA-PH domain of RIAM translocates to the lymphocyte PM much more efficiently than does full-length RIAM. This suggests that the RA-PH domains are inhibited in full-length RIAM. Using truncation mutants, we mapped the autoinhibitory region to the first 57 residues that precede the two N-terminal coiled-coil regions. Because coiled-coil regions are often involved in intra- and intermolecular protein–protein interactions (Mason and Arndt, 2004
), which can be used for autoinhibition of functional domains (Rosenberg et al., 2005
), it is plausible that hetero- or homo-multimerization of RIAM is responsible for obscuring the RA-PH membrane-binding module. If this is the case, then some aspect of inside-out signaling to LFA-1 in lymphocytes must disinhibit the RIAM RA-PH domains. Because talin binds to the RIAM N terminus, it is tempting to speculate that talin binding to RIAM might accomplish this conformational change (). Because talin is also autoinhibited (Goksoy et al., 2008
), it is possible that RIAM and talin work reciprocally to disinhibit each other. Because RIAM binds to both the inner leaflet of the PM and to the β chain of LFA-1 (Kliche et al., 2012
), our model links LFA-1 to talin through membrane-associated RIAM, which would constitute a secondary association in addition to direct binding of talin to the β chain of the integrin (Tadokoro et al., 2003
). However, this model still leaves open the question of what aspect of signaling enables talin binding to RIAM.
Figure 8. Model for RIAM translocation to the PM of lymphocytes. Although Rap1 constitutively associates with multiple cellular membranes by virtue of its geranylgeranyl modification, the relevant pool for LFA-1 regulation is at the PM where it is activated by (more ...)
The immune system can be likened to a double-edged sword. On the one hand, vertebrates cannot survive without it in a world swarming with microbes. On the other hand, it possesses the power to destroy normal tissues. That is, there is a fine balance between host defense and autoimmunity. Lymphocyte adhesion to endothelium and APCs is absolutely required for adaptive immunity and therefore must be tightly controlled. This may explain why RIAM, a molecule which regulates lymphocyte adhesion, is controlled by dual membrane targeting domains that constitute an AND gate rather than a single determinant that would represent a simple switch.